Mucosal Mast Cells Research Articles

Vasopressin augments TNBS-induced colitis through enteric neuronal V1a receptor-mediated COX-2-dependent prostaglandin release from mast cells in mice.

Inflammatory bowel disease (IBD) is a functional disorder with chronic and relapsing clinical features. Vasopressin (VP) is a hormone responsible for water and stress homeostasis and also regulates gastrointestinal inflammation and motility. We explored whether VP was related to IBD pathogenesis and its possible pathway. Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice. The disease activity and colonic damage were evaluated through a scoring system. Locations of the V1a receptor were revealed by immunochemistry method in colon. Ussing chamber technique was performed for the electrophysiological characterization by using rat ileum. The (Arg8 )-Vasopressin (AVP)-evoked short-circuit current (Isc) was recorded in the presence of conivaptan (V1a and V2 receptor antagonist), tolvaptan (V1b receptor antagonist), tetrodotoxin (TTX), atropine, cyclooxygenase (COX) inhibitors (indomethacin, nonspecific COX antagonist; SC560, COX-1 antagonist; NS560, COX-2 antagonist), and a stabilizer of mast cell (cromolyn sodium), respectively. TNBS resulted in the obvious loss of body weight and tissue damages in mice. AVP significantly aggravated the TNBS-induced colitis, which was attenuated by conivaptan but not tolvaptan. V1a receptors were found immunopositive in neurons among the enteric nervous system. AVP evoked a pulsatile response in Isc. Its amplitude, frequency, and cycle duration were around 8-15µA/cm2 , 10-11mHz, and 1.5minutes, respectively. Notably, the AVP-evoked change in Isc was abolished by TTX, atropine, conivaptan, indomethacin, NS560, and cromolyn sodium, respectively. VP-V1a receptor played the proinflammatory role in TNBS-induced colitis by promoting COX-2-dependent prostaglandin release from mucosal mast cells, which was mediated by the cholinergic pathway.

Mucosal Mast Cells Are Increased in the Small Intestine of Patients With Irritable Bowel Syndrome: A Systematic Review and Meta-Analysis

Mucosal Mast Cells Are Increased in the Small Intestine of Patients With Irritable Bowel Syndrome: A Systematic Review and Meta-Analysis

Are Enterocolic Mucosal Mast Cell Aggregates Clinically Relevant in Patients Without Suspected or Established Systemic Mastocytosis?

The World Health Organization considers enterocolic mast cell aggregates with atypical morphologic and/or immunohistochemical features diagnostic of systemic mastocytosis mostly because published data are heavily influenced by inclusion of symptomatic patients with systemic disease. We occasionally encounter atypical mast cells in gastrointestinal biopsy samples from patients in whom systemic mastocytosis is not suspected. The aim of this study was to describe the clinicopathologic features and implications of atypical enterocolic mast cell aggregates in 16 patients without suspected or established systemic mastocytosis. Mast cell infiltrates were assessed for morphology, distribution, associated inflammatory cells, and CD117 and CD25 immunoexpression. Most (63%) patients were women; 15 underwent endoscopic examination for screening (n=7), abdominal pain (n=3), diarrhea (n=3), changing bowel habits (n=1), and dysphagia (n=1). Mast cell aggregates were detected in 1 colectomy specimen for cancer. Colonic involvement was most common (n=14) and resulted in polypoid (n=10), edematous (n=2), or normal (n=3) mucosae. All cases featured CD117/CD25, ovoid mast cells concentrated beneath the epithelium, or diffusely involving the entire mucosal thickness. Eosinophils were numerous and obscured mast cells in 63% of cases. Spontaneous resolution of symptoms occurred in all patients (mean follow-up: 54 mo), and asymptomatic patients remained symptom-free (mean follow-up: 17 mo). Of 4 patients evaluated for systemic mastocytosis, 3 had negative bone marrow biopsies and one lacked a KIT mutation in peripheral blood. We conclude that, although careful clinical assessment of patients with incidental enterocolic mast cell aggregates is reasonable, labeling them with a systemic hematologic disorder may not be justified.

Elevated Tryptase in EoE Is an Independent Phenomenon Associated with Extra-Esophageal Symptoms.

Eosinophilic esophagitis (EoE) is a chronic disease characterized histologically by > 15 eosinophilsper high-power field (eos/hpf). Esophageal mucosal mast cells have been implicated in EoE pathogenesis. The association of atopy with EoE has been established but has not been correlated with levels of serum tryptase. The lack of concurrent atopy in some patients suggests the possibility that atopy may either be the related subtype of EoE or may be a sign of comorbidities. No study has looked at whether patients present with different phenotypes/comorbid disease when they have evidence of elevated serum tryptase. We hypothesized that these patients differ with respect to presentation and comorbidities with more refractory GI disease. To examine whether elevations of serum tryptase associate with different, more severe clinical presentations in EoE patients which may be explained via mast cell activation. Retrospective chart review identified 72 patients with EoE with results for serum tryptase between 2015 and 2016. Patients were classified as TryptaseHI (tryptase > 10.9µg/l) and TryptaseLO (< 10.9µg/l). Clinical characteristics and treatment response were compared using univariate analysis and multivariate regression between the groups. Out of 72 patients, 12 were tested as TryptaseHI (16.7%, 95% CI 8.1-25.3%). TryptaseHI was associated frequently with asthma (P = 0.0003), urticaria (P = 0.002), arthralgia (P = 0.005), sinusitis (P = 0.03), nausea/vomiting (P = 0.046), and eosinophilic gastrointestinal disease (P = 0.001). Asthma and arthralgia were found to be significantly associated with TryptaseHI (P = 0.0013, P = 0.0098, respectively). Mucosal eosinophil counts and tryptase levels were not correlated (R2 0.095, P = 0.77). Tryptase did not resolve with resolution of esophageal eosinophilia. We found that EoE patients with elevated tryptase levels more commonly presented with asthma, urticaria, arthralgia, nausea/vomiting, sinusitis, and more distal eosinophilia. This indicates that atopy in EoE patients warrants further exploration. The lack of correlation between histologic remission and reduction of serum tryptase levels post-treatment suggests that mast cell activation may be an independent, yet associated disease. More study into this unique association is warranted.

Bile acids induce visceral hypersensitivity via mucosal mast cell–to–nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis

Increased colonic bile acid (BA) exposure, frequent in diarrhea-predominant irritable bowel syndrome (IBS-D), can affect gut function. Nerve growth factor (NGF) is implicated in the development of visceral hypersensitivity (VH). In this study, we tested the hypothesis that BAs cause VH via mucosal mast cell (MMC)-to-nociceptor signaling, which involves the farnesoid X receptor (FXR)/NGF/transient receptor potential vanilloid (TRPV)1 axis. BAs were intracolonically administered to rats for 15 d. Visceral sensitivity to colorectal distention and colonic NGF expression were examined. BAs caused VH, an effect that involved MMC-derived NGF and was accompanied by enhanced TRPV1 expression in the dorsal root ganglia. Anti-NGF treatment and TRPV1 antagonism inhibited BA-induced VH. BAs induced NGF mRNA and protein expression and release in cultured mast cells. Colonic supernatants from patients with IBS-D with elevated colonic BA content transcriptionally induced NGF expression. In FXR-/- mice, visceral sensitivity and colonic NGF expression were unaltered after BA treatment. Pharmacological antagonism and FXR silencing suppressed BA-induced NGF expression and release in mast cells. Mitogen-activated protein kinase kinase (MKK) 3/6/p38 MAPK/NF-κB signaling was mechanistically responsible for FXR-mediated NGF expression and secretion. The findings show an MMC-dependent and FXR-mediated pronociceptive effect of BAs and identify the BA/FXR/NGF/TRPV1 axis as a key player in MMC-to-neuron communication during pain processing in IBS.-Li, W.-T., Luo, Q.-Q., Wang, B., Chen, X., Yan, X.-J., Qiu, H.-Y., Chen, S.-L. Bile acids induce visceral hypersensitivity via mucosal mast cell-to-nociceptor signaling that involves the farnesoid X receptor/nerve growth factor/transient receptor potential vanilloid 1 axis.

IL-10 Receptor or TGF-β Neutralization Abrogates the Protective Effect of a Specific Nondigestible Oligosaccharide Mixture in Cow-Milk-Allergic Mice

BackgroundDietary nondigestible, short-chain galacto-, long-chain fructo-, and pectin-derived acidic oligosaccharides (GFAs) lower the effector response in cow-milk-allergic (CMA) mice; and forkhead box P3 (Foxp3)–positive regulatory T cells (Tregs) were shown to contribute to this.ObjectiveThe aim of this study was to assess the contribution of interleukin 10 (IL-10) and transforming growth factor β (TGF-β) to the protective effect of the GFA diet in CMA mice.MethodsFemale C3H/HeOuJ mice, 3–4 wk old, were orally sensitized with cholera toxin (Sham) or whey and cholera toxin (Whey) 1 time/wk for 5 consecutive weeks and challenged with whey 1 wk later. The mice were fed a control or 1% GFA (9:2:1) (Whey+GFA) diet starting 2 wk before the first sensitization. In a second experiment, the mice were also injected with αIL-10 receptor (αIL-10r), αTGF-β, or isotype control antibodies 24 h before each sensitization. The acute allergic skin response, anaphylaxis score, whey-specific IgE, mucosal mast cell protease 1 (mMCP-1), and Treg frequency in the mesenteric lymph nodes (MLNs) and intestinal Foxp3, Il10, and Tgfb mRNA expression were determined.ResultsIn Whey+GFA mice, intestinal Il10, Tgfb, or Foxp3 mRNA expression was 2–10 times higher (P < 0.05) and the MLN Treg frequency was 25% higher compared with Whey mice (P < 0.05). The acute allergic skin response was 50% lower in Whey+GFA mice compared with Whey mice (P < 0.01), and IL-10 receptor (IL-10r) or TGF-β neutralizing antibodies prevented this protective effect (P < 0.001). The Whey mice had higher serum mMCP-1 concentrations and whey–immunoglobulin E (-IgE) levels than Sham mice (P < 0.01), whereas these were not higher in Whey+GFA mice, and neutralizing antibodies partially interfered with these responses.ConclusionsDietary GFAs enhance the Treg frequency in the MLNs and mucosal IL-10 and TGF-β transcription while suppressing the allergic effector response. Neutralizing antibodies showed that the allergy-protective effect of the GFA diet was mediated by IL-10 and TGF-β in CMA mice.

An observational study of headaches in children and adolescents with functional abdominal pain: Relationship to mucosal inflammation and gastrointestinal and somatic symptoms.

Headaches and abdominal pain are among the most common pediatric pain conditions. Mast cells have been implicated in the pathophysiology of migraines, as well as functional dyspepsia (FD) and irritable bowel syndrome (IBS). The primary aims of the current study were to assess headache prevalence in patients with FD and to assess the association between headaches and mucosal mast cells and eosinophils. An additional aim was to explore associations of headache with other symptoms.We conducted a cross-sectional retrospective chart review of 235 consecutive patients with chronic abdominal pain. All patients had completed a standardized questionnaire as part of their routine clinical evaluation. Both gastrointestinal and non-gastrointestinal somatic symptoms were included in the analysis. All patients diagnosed with FD had undergone upper endoscopy with biopsies obtained from the gastric antrum and duodenum and these specimens were utilized to assess eosinophil and mast cell densities, respectively.Overall, 86% of patients fulfilled Rome IV criteria for FD. Headache was reported by 73.8% of FD patients versus 45.2% of non-FD patients (P = .001). Duodenal mast cell densities were significantly increased in those reporting headaches. Headache was not associated with any specific gastrointestinal symptoms but was associated with a wide array of non-gastrointestinal symptoms including fatigue, dizziness, muscle pain, joint pain, and chest pain.Headaches are common in children and adolescents with abdominal pain and, utilizing Rome IV criteria, are specifically associated with FD. In patients with FD, headaches are associated with increased duodenal mast cell density and a variety of somatic symptoms, all of which are possibly the result of mast cell activation.

Changes of gastric mucosal mast cells in patients with functional dyspepsia after Hylicobacter pylori infection

Objective To investigate the changes of gastric mucosal mast cells in patients with functional dyspepsia (FD) after Helicobacter pylori (H.pylori) infection, and to explore the correlation between the changes and dyspeptic symptoms. Methods From November 2012 to March 2014, 32 patients with FD and 16 asymptomatic patients (control group) from Beijing Jishuitan Hospital were enrolled. H. pylori infection situation was examined. The mucosal biopsies from gastric antrum and gastric body were taken for histological examination. The mast cells of gastric mucosa were detected by immunohistochemical stain. T test was performed for statistical analysis. Results Among 32 FD patients, 17 had H. pylori infection (one with gastric body involved, two with gastric antrum involved, 14 with both gastric antrum and gastric body involved), and 15 had no H. pylori infection. Among 16 asymptomatic patients, 12 had H. pylori infection and four had no H. pylori infection. Among FD patients, the percentage of degranulated mast cells in gastric mucosa with H. pylori infection group was (61.45±24.86)%, which was higher than that without H. pylori infection group ((48.34±21.63)%), and the difference was statistically significant (t=-2.145, P=0.036). The percentage of degranulated mast cells in gastric mucosa with H. pylori infection group was (29.73±12.99)%, which was higher than that without H. pylori infection group ((20.77±10.86)%), and the difference was statistically significant (t=-2.856, P=0.006). In H. pylori infection group, the percentage of degranulated mast cells in gastric body mucosa of FD patients was (66.99±18.85)%, which was higher than that of asymptomatic patients in control group ((46.77±8.43)%), and the difference was statistically significant (t=3.642, P=0.002). The ratio of degranulated mast cells in gastric body mucosa and the density of mast cells in gastric antrum mucosa of prandial distress syndrome (PDS) patients, the density of mast cells in gastric body mucosa of epigastric pain syndrome (EPS) patients and the ratio of degranulated mast cells in gastric body mucosa and the degree of degranulation of mast cells of PDS and EPS patients were all higher than those of asymptomatic patients in control group, and the differences were statistically significant (t=2.750, 3.601, 2.799, 9.522 and 5.755, all P<0.05). Conclusions The dyspeptic symptoms of FD patients with H. pylori infection is correlated with the changes of mast cells. With the infection of H. pylori, dyspeptic symptoms of different subtypes of FD may be correlated with the changes of mast cells in different gastric region. H. pylori infection may cause the dyspeptic symptoms of FD patients by increase the number and activation of mast cells. Key words: Helicobacter pylori; Mast cells; Functional dyspepsia; Dyspeptic symptom

Hepatic Mitochondrial Dysfunction and Immune Response in a Murine Model of Peanut Allergy.

Background: Evidence suggests a relevant role for liver and mitochondrial dysfunction in allergic disease. However, the role of hepatic mitochondrial function in food allergy is largely unknown. We aimed to investigate hepatic mitochondrial dysfunction in a murine model of peanut allergy. Methods: Three-week-old C3H/HeOuJ mice were sensitized by the oral route with peanut-extract (PNT). We investigated: 1. the occurrence of effective sensitization to PNT by analysing acute allergic skin response, anaphylactic symptoms score, body temperature, serum mucosal mast cell protease-1 (mMCP-1) and anti-PNT immunoglobulin E (IgE) levels; 2. hepatic involvement by analysing interleukin (IL)-4, IL-5, IL-13, IL-10 and IFN-γ mRNA expression; 3. hepatic mitochondrial oxidation rates and efficiency by polarography, and hydrogen peroxide (H2O2) yield, aconitase and superoxide dysmutase activities by spectrophotometry. Results: Sensitization to PNT was demonstrated by acute allergic skin response, anaphylactic symptoms score, body temperature decrease, serum mMCP-1 and anti-peanut IgE levels. Liver involvement was demonstrated by a significant increase of hepatic Th2 cytokines (IL-4, IL-5 and IL-13) mRNA expression. Mitochondrial dysfunction was demonstrated by lower state 3 respiration rate in the presence of succinate, decreased fatty acid oxidation in the presence of palmitoyl-carnitine, increased yield of ROS proven by the inactivation of aconitase enzyme and higher H2O2 mitochondrial release. Conclusions: We provide evidence of hepatic mitochondrial dysfunction in a murine model of peanut allergy. These data could open the way to the identification of new mitochondrial targets for innovative preventive and therapeutic strategies against food allergy.

Interleukin-9 promotes early mast cell-mediated expulsion of Strongyloides ratti but is dispensable for generation of protective memory

IL-9 is a cytokine with pleiotropic function that mediates allergic inflammation and immunity to intestinal helminth parasites. Accumulating evidence suggests that IL-9 acts via both, initiation and regulation of adaptive immune responses and direct activation of intestinal effector pathways. Here we use IL-9 receptor deficient mice on BALB/c and C57BL/6 genetic background to dissect effector and regulatory functions of IL-9 during infection with the parasitic nematode Strongyloides ratti. IL-9 receptor-deficient mice displayed increased intestinal parasite burden and prolonged infection irrespective of the genetic background of the mice. Increased parasite burden was correlated to a reciprocally reduced early degranulation of mucosal mast cells, reduced intestinal IL-13 expression and caused by IL-9 receptor deficiency on hematopoietic cells. We observed additional significant changes in the adaptive immune response to S. ratti infection in the absence of the IL-9 receptor that depended on the mouse strain. However, the generation of protective memory to a second infection was intact in IL-9 receptor-deficient mice, irrespective of the genetic background. In summary, our results support a central role for IL-9 as an early mast cell activating effector cytokine during intestinal helminth infection while non-redundant functions in the initiation and amplification of adaptive immune responses were not apparent.

Characterization of the phenotype of the connective tissue mast cell knockout Mcpt5Cre + ;Rosa(DTA) mice in the lung

Abstract Mast cells are roughly grouped as resident connective tissue mast cells (CTMCs) and mucosal mast cells which are recruited to mucosal epithelia during inflammation. Although mast cells are known to play critical roles in allergic lung inflammation, little is known about the specific function of pulmonary CTMCs under any condition. To investigate their function, we employed Mcpt5Cre+; Rosa(DTA) transgenic mice to eliminate cells that ever expressed Mcpt5, a classic marker of CTMCs. Unexpectedly, we discovered that the lungs of these mice contained an increased number of mast cells, likely mucosal mast cells. In addition, we found spontaneous inflammation characterized by recruitment of neutrophils and eosinophils, elevated il-33 expression, mucus overproduction and excessive collagen in the airway. Inflammation was detected as early as postnatal day 21 but resolved around 2–3 months of age. In contrast, other tissues that normally have abundant CTMCs, such as the skin and intestine, present no baseline phenotypes in Mcpt5Cre+;Rosa(DTA) mice. These findings suggest a tissue-specific role of CTMCs in maintaining tolerance in the lung, potentially by inhibiting the recruitment of pro-inflammatory mucosal mast cells. Further, in conflict with previous findings that the Mcpt5Cre line specifically targets CTMCs, lineage labeling using Mcpt5Cre+;Rosa(TmRed) mice revealed the presence of TmRed reporter in CTMCs and a small subset of other leukocytes in the lung. While these leukocytes are being mapped, the already identified cell types are unlikely to contribute to the baseline phenotype of Mcpt5Cre+;Rosa(TmRed) mice. Our ongoing studies aim to determine the role of CTMCs in establishing immune tolerance.

IL-9 and Mast Cells Are Key Players of Candida albicans Commensalism and Pathogenesis in the Gut

SummaryCandida albicans is implicated in intestinal diseases. Identifying host signatures that discriminate between the pathogenic versus commensal nature of this human commensal is clinically relevant. In the present study, we identify IL-9 and mast cells (MCs) as key players of Candida commensalism and pathogenicity. By inducing TGF-β in stromal MCs, IL-9 pivotally contributes to mucosal immune tolerance via the indoleamine 2,3-dioxygenase enzyme. However, Candida-driven IL-9 and mucosal MCs also contribute to barrier function loss, dissemination, and inflammation in experimental leaky gut models and are upregulated in patients with celiac disease. Inflammatory dysbiosis occurs with IL-9 and MC deficiency, indicating that the activity of IL-9 and MCs may go beyond host immunity to include regulation of the microbiota. Thus, the output of the IL-9/MC axis is highly contextual during Candida colonization and reveals how host immunity and the microbiota finely tune Candida behavior in the gut.

Sa1109 - Relationship Between Esophageal Mucosal Innervation and Mast Cell Infiltration in Patients with Non Erosive Reflux Disease (Nerd)

Sa1109 - Relationship Between Esophageal Mucosal Innervation and Mast Cell Infiltration in Patients with Non Erosive Reflux Disease (Nerd)

IL-4 signaling regulates the IL-9 producing mast cells (MMC9) gene expression and function in food-induced anaphylaxis in mice

Abstract We have recently described an IL-9 producing mucosal mast cell population (MMC9s) that drives intestinal mastocytosis and food-induced anaphylaxis. The cytokine signaling pathways involved in the regulation of MMC9 function in food allergic reactions are currently not well understood. Employing a murine model of food-induced anaphylaxis and WT, gain-of-function Il4RaF709mice, and Il4Rα−/− mice we revealed an important role for IL-4 signaling in the enhancement of MMC9 frequency and this positively correlated with elevated serum levels of MCPT-1, CD4+TH2 cells, intestinal mastocytosis and increased susceptibility to food-induced anaphylaxis. Employing an adoptive transfer model of food allergy, we show direct IL-4 signaling is required for increased levels of MMC9 and development of food-induced anaphylaxis. RNA-seq analysis of purified WT, Il4RaF709and Il4Rα−/−MMC9s identified that IL-4-signaling pathway differentially regulates 238 genes that were identified to be associated with inflammatory responses and cytokine signaling processes by GO network analyses. Functional analysis revealed that IL-4 upregulates IL-9 and not IL-13 production in MMC9s. These results suggest that IL-4 signaling directly regulates MMC9 gene expression and function and onset of food-induced anaphylaxis. These studies suggest that targeting MMC9s through IL-4 signaling blockade maybe therapeutically beneficial for prevention and treatment of food allergy.

An adjuvant-free mouse model of anaphylaxis to saline-soluble wheat protein

Abstract Wheat is a major food that can trigger life-threatening systemic anaphylaxis. Mechanism of sensitization to wheat anaphylaxis is not completely understood at present. For example, whether or not skin exposure to saline-soluble wheat protein (SSWP) can cause sensitization is unknown. Here we tested the hypothesis that transdermal exposure (TDE) to SSWP from durum wheat results in IgE response and clinical sensitization for anaphylaxis. Groups of Balb/c mice weaned onto a plant free diet received six TDEs with SSWP (1 mg/mouse/week) or saline over a 6 week period without adjuvant. Blood was analyzed for IgE responses by ELISA, and anaphylaxis and mucosal mast cell protease (mMCP-1) release upon challenge were quantified. Spleen tissue was analyzed for cytokines using a protein microarray system. Results showed significant IgE response, hypothermia shock response and elevation of mMCP-1 upon challenge. Spleen analysis showed significant elevations of IL-4/5/7/10/17B,E,F, and TSLP and significant reductions of IL-1a and IL-6. The following cytokines were not significantly altered: IL-1b/2/9/13/17A/22/23, IFNg and TNFa.

Mast cell-driven clearance in N. brasiliensis is masked by IL-25-dependent B1 cell IgE.

Abstract A large amount of poly-specific IgE is generated during helminth infection. We have previously shown that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasite Nippostrongylus brasiliensis. In vitro analysis demonstrated a novel role for IL-25-enhancement of IgE in B1 cells from helminth infected mice. In T cell-reconstituted RAG1−/− mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. IL-25 receptor-deficient B1 cells did not impede this clearance. Mucosal mast cells mediated the B2 cell-enhancement of clearance only in the absence of B1 cells. This was shown using mast cell depletion with anti-ckit (ACK.2) in a helminth-infected RAG1−/− reconstitution model. Additionally, gene expression analysis of the jejunum on day 7 post-inoculation found increased mast cell protease genes and mucus related genes in RAG1−/− mice reconstituted with B2 cells over T cells alone. This revealed a potential mechanism for B2 cell-enhanced clearance. Finally, R1E4, an antibody that prevents IgE from binding to FcɛRI, was administered in a model of RAG1−/− reconstitution infected with helminth and results confirmed that the B2 cell IgE/mast cell clearance enhancement was mediated through FcɛRI. Overall, these data shine new light on B2 cell derived IgE and mast cells and their role in helminth clearance that is impeded by IL-25-driven B1 cell IgE.

Ovarian mast cells migrate toward ovary-fimbria connection in neonatal MRL/MpJ mice.

MRL/MpJ mice have abundant ovarian mast cells (MCs) as compared with other strains at postnatal day 0 (P0); however, they sharply decrease after birth. These ovarian MCs, particularly beneath the ovarian surface epithelium (SE), which express mucosal MC (MMC) marker, might participate in early follicular development. This study investigated the changes in spatiotemporal distribution of MCs in the perinatal MRL/MpJ mouse ovaries. At P0 to P7, the MCs were densely localized to the ovary, especially their caudomedial region around the ovary-fimbria connection. The neonatal ovarian MCs showed intermediate characteristics of MMC and connective tissue MC (CTMC), and the latter phenotype became evident with aging. However, the expression ratio of the MMC to CTMC marker increased from P0 to P4 in the MRL/MpJ mouse ovary. Similarly, the ratio of MCs facing SE to total MC number increased with aging, although the number of ovarian MCs decreased, indicating the relative increase in MMC phenotypes in the early neonatal ovary. Neither proliferating nor apoptotic MCs were found in the MRL/MpJ mouse ovaries. The parenchymal cells surrounding MCs at ovary-fimbria connection showed similar molecular expression patterns (E-cadherin+/Foxl2-/Gata4+) as that of the ovarian surface epithelial cells. At P2, around the ovary-fimbria connection, c-kit- immature oocytes formed clusters called nests, and some MCs localized adjacent to c-kit- oocytes within the nests. These results indicated that in postnatal MRL/MpJ mice, ovarian MCs changed their distribution by migrating toward the parenchymal cells composing ovary-fimbria connection, which possessed similar characteristics to the ovarian surface epithelium. Thus, we elucidated the spatiotemporal alterations of the ovarian MCs in MRL/MpJ mice, and suggested their importance during the early follicular development by migrating toward the ovary-fimbria connection. MRL/MpJ mice would be useful to elucidate the relationship between neonatal immunity and reproductive systems.

Corticotrophin Releasing Hormone Regulates NLRP6 and Disrupts Mucosal Homeostasis in Functional Dyspepsia

BackgroundFunctional dyspepsia (FD) affects 15% of the population and is characterised by recurring upper gastrointestinal (GI) symptoms occurring in the absence of clinically identifiable pathology. The pathophysiology of FD is poorly understood, however recent work has demonstrated increased numbers of mucosal mast cells and eosinophils in patients with FD. Psychological stress is a key factor associated with the onset of FD and is a trigger of symptom exacerbations, however the biological mechanisms through which stress may lead to FD symptoms have not yet been defined. Recent pre‐clinical work identified that psychological stress downregulates the innate epithelial immune protein NLRP6. NLRP6 has been shown to regulate GI mucus secretion and is responsible for activation of Interleukin 18 (IL‐18). IL‐18 is potently chemotactic for mast cells and eosinophils. We hypothesised that stress hormone levels are altered in patients with FD, leading to alterations in GI mucosal homeostasis and immune cell recruitment.MethodsGoblet cell‐like HT29‐MTX‐E12 (E12) cells were grown on permeable membranes over 21 days to form monolayers with apical‐basolateral polarity. Cells were treated basolaterally with the stress hormone corticotrophin releasing hormone (CRH) (0.05μM or 0.5μM) for 16 hours. mRNA was extracted for qPCR analysis and immunoblots were performed on whole protein lysates. For mucous‐layer analysis, monolayers were snap frozen and stained with alcian blue. FD patients were identified by the Rome III criteria; asymptomatic individuals were recruited as controls. Serum CRH levels were measured by ELISA. Pinch biopsies were collected from the duodenum of FD patients and non‐FD controls. Biopsies were either formalin fixed and stained immunohistochemically or mRNA was extracted from enriched epithelial cell preparations for qPCR analysis.ResultsNLRP6 expression was increased in monolayers treated with 0.05μM CRH and decreased in monolayers treated with 0.5μM CRH (n=3, p=0.02), suggesting a dynamic regulation. CRH treatment at both 0.05μM and 0.5μM significantly increased mucus secretion in monolayers (n=3, p=0.05 and p&lt;0.0001 respectively). Levels of CRH were significantly reduced in both duodenal tissue (n=6, p=0.001) and serum (n=10, p=0.013) of FD patients compared to non‐FD controls. Duodenal NLRP6 was significantly increased in patients with FD compared to non‐FD controls (n=6, p=0.007) and epithelial expression of NLRP6 was positively correlated with expression of IL‐18 (r=0.95, p=0.015) in FD patients, but not non‐FD controls.ConclusionThis study has identified that CRH dynamically regulates NLRP6 in vitro and stimulates mucus secretion. Therefore, the concurrent decrease in CRH and increase in NLRP6 observed in FD patients provides a potential pathway through which psychological stress may lead to disruption of GI mucosal homeostasis and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Eosinophils and Mast Cells in Functional Dyspepsia: an Ultrastructural Evaluation

We recently identified mucosal mast cell and eosinophil hyperplasia in association with a duodenal impaired barrier function in functional dyspepsia (FD). We aimed to further describe the implication of these immune cells by assessing their activation state at the ultrastructural level and by evaluating the association between impaired epithelial integrity and immune activation. Duodenal biopsies were obtained from 24 FD patients and 37 healthy controls. The ultrastructure of mast cells and eosinophils was analyzed by transmission electron microscopy. Transepithelial electrical resistance and paracellular permeability were measured to evaluate epithelial barrier function. The type of degranulation in eosinophils and mast cells was piecemeal. Eosinophils displayed higher degree of degranulation in FD patients than in controls (p < 0.0001). Quantification revealed a decreased granular density in eosinophils of FD patients (p < 0.0001). The degree of degranulation in mast cells was similar in both groups. However, a more heterogeneous profile was found in the FD group (p < 0.0001). No association between epithelial integrity and the number and activation state of mucosal eosinophils and mast cells was found. We demonstrated ultrastructural changes in degranulation state of eosinophils and mast cells, suggesting that eosinophil and mast cell activation play a role in the pathophysiology of FD.

IL-33/ST2 signalling and crosstalk with Fc\u03b5RI and TLR4 is targeted by the parasitic worm product, ES-62

ES-62 is a secreted parasitic worm-derived immunomodulator that exhibits therapeutic potential in allergy by downregulating aberrant MyD88 signalling to normalise the inflammatory phenotype and mast cell responses. IL-33 plays an important role in driving mast cell responses and promoting type-2 allergic inflammation, particularly with respect to asthma, via MyD88-integrated crosstalk amongst the IL-33 receptor (ST2), TLR4 and FcεRI. We have now investigated whether ES-62 targets this pathogenic network by subverting ST2-signalling, specifically by characterising how the functional outcomes of crosstalk amongst ST2, TLR4 and FcεRI are modulated by the worm product in wild type and ST2-deficient mast cells. This analysis showed that whilst ES-62 inhibits IL-33/ST2 signalling, the precise functional modulation observed varies with receptor usage and/or mast cell phenotype. Thus, whilst ES-62’s harnessing of the capacity of ST2 to sequester MyD88 appears sufficient to mediate its inhibitory effects in peritoneal-derived serosal mast cells, downregulation of MyD88 expression appears to be required to dampen the higher levels of cytokine production typically released by bone marrow-derived mucosal mast cells.