Mucosal Mast Cells Research Articles

Mucosal Mast Cell Distribution in the Gastrointestinal Tract of Children: A Preliminary Study for Establishing Reference Values.

The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed. Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects, a chronic inflammatory disease (eg, inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria. There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ± 4.0/ high power field [HPF] vs 32.0 ± 10.1/HPF; P = 0.034) and ascending colon (44.8 ± 10.4/HPF vs 60.4 ± 24.3/HPF; P = 0.047). Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated.

Duodenal Mucosal Barrier in Functional Dyspepsia

In addition to gastric sensorimotor dysfunctions, functional dyspepsia (FD) is also variably associated with duodenal micro-inflammation and epithelial barrier dysfunction, the pathogenesis and clinical significance of which are unknown. Our hypothesis was that miRNAs and/or inflammation degrade epithelial barrier proteins, resulting in increased duodenal mucosal permeability in FD. We compared the duodenal mucosal gene expression and miRNAs, invivo permeability (lactulose-mannitol excretion between 0 and 60 and 60 and 120 minutes after saccharide ingestion), exvivo assessments (transmucosal resistance, fluorescein isothiocyanate [FITC]-dextran flux, and basal ion transport), and duodenal histology (light and electron microscopy) in 40 patients with FD and 24 controls. Compared with controls, the mRNA expression of several barrier proteins (zonula occludens-1, occludin, claudin-12, and E-cadherin) was modestly reduced (ie, a fold change of 0.8-0.85) in FD with increased expression of several miRNAs (eg, miR-142-3p and miR-144-3-p), which suppress these genes. The urinary lactulose excretion and the lactulose:mannitol ratio between 60 and 120 minutes were greater in FD than in controls (P < .05). The FITC-dextran flux, which reflects paracellular permeability, was inversely correlated (r= -0.32, P= .03) with transmucosal resistance and directly correlated (r= 0.4, P= .02) with lactulose:mannitol ratio. Other parameters (mucosal eosinophils, intraepithelial lymphocytes, and mast cells, transmucosal resistance, FITC-dextran flux, average intercellular distance, and proportion of dilated junctions) were not significantly different between groups. In FD, there is a modest reduction in the expression of several duodenal epithelial barrier proteins, which may be secondary to up-regulation of regulatory miRNAs, and increased small intestinal permeability measured invivo.

Sex differences in anatomic plasticity of gut neuronal-mast cell interactions.

The gut wall houses mast cells that are anatomically situated near enteric neuronal fibers. Roles of specific neuropeptides in modulating function of immune components like mast cells in response to challenge with bacterial components are relatively unknown. Investigating such interactions requires models that include diverse cellular elements in native anatomic arrangements. Using an organotypic slice model that maintains gut wall cellular diversity ex vivo, the present study compared responses between tissues derived from male and female mice to examine neural‐immune signaling in the gut wall after selected treatments. Ileum slices were treated with pharmacological reagents that block neuronal function (e.g., tetrodotoxin) or vasoactive intestinal peptide (VIP) receptors prior to challenge with lipopolysaccharide (LPS) to assess their influence on anatomic plasticity of VIP fibers and activation of mast cells. Sex differences were observed in the number of mucosal mast cells (c‐kit/ACK2 immunoreactive) at baseline, regardless of treatment, with female ileum tissue having 46% more ACK2‐IR mast cells than males. After challenge with LPS, male mast cell counts rose to female levels. Furthermore, sex differences were observed in the percentage of ACK2‐IR cells within 1 µm of a VIP+ neuronal fiber, and mast cell size, a metric previously tied to activation, with females having larger cells at baseline. Male mast cell sizes reached female levels after LPS challenge. This study suggests sex differences in neural‐immune plasticity and in mast cell activation both basally and in response to challenge with LPS. These sex differences could potentially impact functional neuroimmune response to pathogens.

Modern Imaging Technologies of Mast Cells for Biology and Medicine (Review).

Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is evidence for their contributing to the pathogenesis of a number of atopic, autoimmune, as well as cardiovascular, oncologic, neurologic, and other diseases (allergy, asthma, eczema, rhinitis, anaphylaxis, mastocytosis, multiple sclerosis, rheumatoid arthritis, inflammatory gastrointestinal and pulmonary diseases, migraine, etc.). The diagnosis of many diseases and the study of mast cell functions in health and disease require their identification; so, the knowledge on adequate imaging techniques for mast cells in humans and different species of animals is of particular importance.The present review summarizes the data on major methods of mast cell imaging: enzyme histochemistry, immunohistochemistry, as well as histochemistry using histological stains. The main histological stains bind to heparin and other acidic mucopolysaccharides contained in mast cells and stain them metachromatically. Among these are toluidine blue, methylene blue (including that contained in May-Grünwald–Giemsa stain), thionin, pinacyanol, and others. Safranin and fluorescent dyes: berberine and avidin — also bind to heparin. Longer staining with histological dyes or alcian blue staining is needed to label mucosal and immature mast cells.Advanced techniques — enzyme histochemistry and especially immunohistochemistry — enable to detect mast cells high-selectively using a reaction to tryptases and chymases (specific proteases of these cells). In the immunohistochemical study of tryptases and chymases, species-specific differences in the distribution of the proteases in mast cells of humans and animals should be taken into account for their adequate detection. The immunohistochemical reaction to immunoglobulin E receptor (FcεRI) and c-kit receptor is not specific to mast cells, although the latter is important to demonstrate their proliferation in normal and malignant growth.Correct fixation of biological material is also discussed in the review as it is of great significance for histochemical and immunohistochemical mast cell detection.Fluorescent methods of immunohistochemistry and a multimarker analysis in combination with confocal microscopy are reported to be new technological approaches currently used to study various mast cell populations.

Pathophysiological Roles of Neuro-Immune Interactions between Enteric Neurons and Mucosal Mast Cells in the Gut of Food Allergy Mice.

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

Effects of analgesic and surgical modality on immune response in colorectal cancer surgery

Background and objectiveDifferent surgical methods, anesthesia, and analgesia are known to modify the surgical stress response, especially in patients with malignancy. We compared the impact of patient-controlled intravenous (PCA) versus epidural analgesia (EDA) on tumor-related mucosal immune response in patients undergoing open or laparoscopic surgery for colorectal cancer. MethodsIn a University Hospital subgroup (n = 43) of a larger cohort (n = 235) of patients undergoing open or laparoscopic surgery for colorectal carcinoma randomized to PCA or EDA, colorectal tissues were stained for interleukin-10 (IL-10), tumor necrosis factor (TNF), and mast cell tryptase and then examined by immunofluorescence microscopy. ResultsMore IL-10+-cells were found in patients undergoing open compared to laparoscopic surgery in the PCA (P < 0.05) and EDA group (P < 0.0005), respectively, and numbers of TNF+-cells were higher in the open surgery group who received PCA (P < 0.05). No differences in IL-10 or TNF expressions were detected between EDA/PCA within the open or laparoscopic surgery groups, respectively. Fewer mast cells were observed in patients undergoing laparoscopic compared to open surgery combined with PCA (P < 0.05). Within the open surgery group, EDA resulted in fewer mucosal mast cells compared to the PCA group (P < 0.05). ConclusionsThe surgical method, rather than type of analgesia, may have higher impact on peri-operative inflammation. Laparoscopic surgery when combined with EDA for colorectal cancer caused a decrease in the TNF and IL-10 expression and mast cells. EDA seems to have an anti-inflammatory effect on cancer-related inflammation during open surgery.

Sex Differences in Neural – Immune – Epithelial Anatomical Plasticity in the Mouse Small Intestine

Background Pathways of neural signaling leading to alterations in gut wall immune – epithelial function are not well understood, particularly in the context of sex. Specific neural factors regulating epithelial or immune cell localization and secretion are emerging. Vasoactive intestinal peptide (VIP), often a neuropeptide, can play a role in goblet cell production (1) and signals bi-directionally with enteric mast cells (2). Immune components like mast cells are key in gut wall defense against pathogens. Epithelial components of host defense include goblet cells secreting mucus to form a physical barrier, and Paneth cells secreting anti-microbial compounds. Neural fibers course intimately with goblet and Paneth cells and may signal with gut mucosal mast cells (2). The present study establishes the anatomic arrangement of immunoreactive VIP (VIP-IR) fibers in relation to mast and Paneth cells in the mouse ileum, and tests the hypothesis that sex is a key variable in neural – immune – epithelial signaling in the gut wall in healthy and perturbed tissue. Methods We used an organotypic gut slice model that maintains the cellular diversity of the gut wall ex vivo. Slices were treated with selected pharmacological reagents that block neuronal function (e.g., tetrodotoxin) or VIP receptors prior to challenge with lipopolysaccharide (LPS) to assess their influence on anatomic plasticity of VIP-IR fibers with Paneth cells and mast cells at 48h ex vivo. Results There were more mast cells in females than males whether drop fixed in vivo (214%) or after 48h of ex vivo culture (53%). The mast cells in tissue from females drop fixed in vivo were ~18% larger than in males, and similarly ~26% larger in slices of ileum maintained ex vivo for 48h (Fig 1A-D). Approximately 70% of mast cells had VIP-IR fibers within 1µm, regardless of sex. However, those mast cells within 1 µm of a fiber were 15% larger than those without a nearby fiber, regardless of sex (Fig 1E-H). A small percentage of mast cells were induced to show substantial VIP-IR ex vivo only in slices treated with a VIP receptor antagonist or tetrodotoxin. Surprisingly, this effect occurred more in female slices (~200% more) than in male (Fig 1I-L). Paneth cells were labelled with the lectin Ulex europaeusagglutinin-1 and >75% were within 1 µm of a VIP-IR neuronal fiber, independent of sex or treatment with LPS. By contrast, Paneth cells in slices from females showed 51% greater fluorescent intensity than males, a difference not found after LPS treatment (Fig 2). Conclusions The present study demonstrates a close anatomical relationship of mast and Paneth cells with VIP-IR neuronal fibers. We extend previous work demonstrating a functional influence of VIP over goblet cell production in mouse ileum. The current results suggest sex differences in neural-immune and neural-epithelial plasticity that could potentially impact function. Additional data is needed to test the hypothesis that VIP has a functional role in regulating mast and Paneth cell secretion of histamine and anti-microbials in healthy and pathogen challenged states. References (1) Schwerdtfeger LA and Tobet SA. Physiol Rep 8:e14363, 2020. (2) Buhner S et al., Front Physiol 8:971, 2017.

The role of the stress‐activated transcription factor Nrf2 in OVA‐induced food allergy

Immune-mediated adverse reactions to food allergens are rising at a striking rate, but the underlying cause is unknown. The purpose of the present studies was to determine the effect of the food preservative tert-butylhydroquinone (tBHQ) on anaphylaxis in a mouse model of food allergy. Our preliminary studies demonstrate that skin-sensitization with OVA induced a markedly higher OVA-specific IgE response in mice on a tBHQ diet. Likewise, in response to OVA challenge by the oral route, a greater decrease in body temperature during anaphylaxis was observed in the tBHQ-treated group. Previously published in vitro studies suggest that tBHQ promotes Th2 polarization, a key step in the development of allergy, through activation of the stress-activated transcription factor, Nrf2. Therefore, we proposed the hypothesis that tBHQ exacerbates allergic response in ovalbumin (OVA) sensitized mice through activation of Nrf2. We implemented adoptive transfers of CD4 T cells from wild-type or Nrf2-null mice into immunodeficient SCID mice (all mice also received wild-type B cells). The study revealed marked genotype differences. Notably, dietary tBHQ had little effect on food allergy in recipients of Nrf2-deficient CD4 T cells. These mice showed markedly attenuated sensitization as compared to mice who received wild-type CD4 T cells. Strikingly, mice receiving Nrf2-deficient T cells did not become anaphylactic following OVA challenge. In contrast, tBHQ caused an increase in OVA-IgE plasma levels and a greater drop in body temperature in mice receiving wild-type CD4 T cells, suggesting these effects are Nrf2 dependent. In addition, recipients of Nrf2-deficient CD4 T cells showed a greatly attenuated percentage of Th2 cells and mucosal mast cell degranulation response, following OVA challenge. Taken together, these data suggest a critical role for Nrf2 during food allergen-induced sensitization and anaphylaxis and demonstrate that tBHQ exacerbates food allergy in a CD4 T cell- Nrf2 dependent manner in mice.

Mucosal eosinophils, mast cells, and intraepithelial lymphocytes in youth with rumination syndrome.

Rumination syndrome has been associated with increased duodenal eosinophils and intraepithelial lymphocytes in adults. The aims of the current study were to assess densities of antroduodenal eosinophils and mast cells and duodenal intraepithelial lymphocytes in youth with rumination syndrome and to compare cell densities in those with and without abdominal pain or early satiety. Twenty-eight youth fulfilling Rome IV criteria for rumination syndrome who had undergone endoscopy were identified and compared to 10 controls. Antral and duodenal biopsies were assessed to determine densities of eosinophils, mast cells, and intraepithelial lymphocytes. Cell densities were also compared between rumination patients with and without abdominal pain and those with and without early satiety. Antral mast cell (peak 18.5±6.5 vs. 12.5±2.7) and eosinophil (peak 9.6±5.2 vs. 4.9±2.1) densities were significantly greater in patients with rumination syndrome as compared to controls. Duodenal intraepithelial lymphocyte densities were also increased in rumination syndrome (18.9 ± 5.1 vs. 11.7 ± 1.5; p<.001). Associations were independent of the presence of abdominal pain or early satiety. In conclusion, we found an increase in eosinophil and mast cell densities in the gastric antrum and an increase in intraepithelial lymphocytes in the duodenum in youth with rumination syndrome which was independent of the presence of abdominal pain or early satiety. These findings suggest a potential role for inflammation in the pathophysiology of rumination syndrome. Future studies should address whether treatment directed at these cells are beneficial in treating rumination syndrome.

Piper nigrum extract attenuates food allergy by decreasing Th2 cell response and regulating the Th17/Treg balance.

Piper nigrum is extensively utilized because of its antioxidation, antiallergic, antitumor, antiinflammatory, antidiarrhea, and gastrointestinal protection. We attempted to indicate whether the Piper nigrum extract (PNE) could alleviate ovalbumin (OVA)-induced food allergy, and to explore its potential mechanism. An OVA-induced food allergy mouse model was established, and different concentrations of PNE were administrated. Symptoms of food allergy, levels of immunoglobulin E (IgE), mucosal mast cell protease-1 (mMCP-1), and intestine pathological changes were assessed. Additionally, the expressions of T helper (Th) 2, Th17 and regulatory T (Treg)-associated cytokines and the proportion of Th17 and Treg cells in CD4+ T cells were measured. We found PNE attenuated symptoms of food allergy and decreased the levels of IgE and mMCP-1. In PNE group, the infiltration degree of inflammatory cells was ameliorated and the villi of small intestine were more complete. Moreover, the expressions of Th2 and Th17 cell-associated cytokines were down-regulated by PNE pretreatment, while the levels of Treg cell-associated cytokines were up-regulated. PNE decreased the number of Th17 cells, while increased the Tregs cells. PNE treatment dose-dependently improved the Th17/Treg balance. PNE plays a protective role in OVA-induced food allergy through inhibiting Th2 cell response and regulating the Th17/Treg balance.

Elucidating different pattern of immunoregulation in BALB/c and C57BL/6 mice and their F1 progeny

Helminths are large multicellular parasites that infect one quarter of the human population. To prolong their survival, helminths suppress the immune responses of their hosts. Strongyloides ratti delays its expulsion from the gut by induction of regulatory circuits in a mouse strain-specific manner: depletion of Foxp3+ regulatory T cells (Treg) improves the anti-S. ratti immunity in BALB/c but not in C57BL/6 mice. In the current study we compare the hierarchy of immunoregulatory pathways in BALB/c, C57BL/6 mice and their F1 progeny (BALB/c × C57BL/6). Using multicolor flow cytometry, we show that S. ratti induces a distinct pattern of inhibitory checkpoint receptors by Foxp3+ Treg and Foxp3− T cells. Intensity of expression was highest in C57BL/6 and lowest in BALB/c mice, while the F1 cross had an intermediate phenotype or resembled BALB/c mice. Treg subsets expanded during infection in all three mouse strains. Similar to BALB/c mice, depletion of Treg reduced intestinal parasite burden and increased mucosal mast cell activation in S. ratti-infected F1 mice. Our data indicate that Treg dominate the regulation of immune responses in BALB/c and F1 mice, while multiple regulatory layers exist in C57BL/6 mice that may compensate for the absence of Treg.

SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Thymic mast cells: From morphology to physiology

This minireview summarizes some of our results on the structure, origin, and functions of thymic mast cells, as well as their role in stress-induced thymic atrophy. A comparison of the thymic mast cells with the connective tissue and mucosa mast cells is carried out. The morphological and cytochemical similarity of thymic and mucosa mast cells has been proven. The mechanisms of neuro-mast cell interaction, as well as the features of intercellular signaling are analyzed. Another aspect of the review is related to the assessment of thymic mast cell functions in stress-induced atrophy. It is hypothesized that the main function of these cells under stress is to regulate the T-cell emigration. The role of the thymus as an endocrine gland is also discussed. Probably, its endocrine function is mainly associated with the thymic mast cells which are regulated by the nervous system. Regardless of their localization and despite their hemopoietic origin, all mast cells are an important addition to the nervous system. They are a unique combination of both receptor and effector cells located in all organs and tissues. The mast cells supply the central nervous system with information about any local changes in tissue homeostasis and enhance local nervous influences. At the same time, mast cells are extremely important for neuro-immune communication as well as their mutual control on local defense reactions and mucosa conditions.

Identification of key pathways and gene expression in the activation of mast cells via calcium flux using bioinformatics analysis

Mast cells are the main effector cells in IgE-associated allergic disorders, and we have reported that mucosal mast cells (MMCs) play a more important role in the development of food allergy (FA). IgE with antigen or calcium ionophore stimulation can lead to the activation of MMCs via a calcium-dependent pathway. The purpose of the present study was to identify gene signatures with IgE/antigen (dinitrophenyl-bovine serum albumin, DNP-BSA) or calcium ionophore (A23187) on the activation of MMCs. Differentially expressed genes between the two types of samples were identified with microarray analysis. Gene ontology functional and pathway enrichment analyses of differentially expressed genes were performed using the database for annotation, visualization, and integrated discovery software. The results showed that IgE/antigen and A23187 could induce degranulation, increase vacuoles, and elevate the cytosolic calcium concentration in MMCs. Furthermore, GeneChip analysis showed that the same 134 mRNAs were altered with IgE/DNP-BSA and A23187, suggesting that DNP-BSA/IgE and A23187 affect the same signal pathway partly in degranulation. KEGG analysis showed that the data were enriched in NF-κB, TNF, MAPK, transcription factor activity, DNA binding, and nucleic acid binding, suggesting that activation of MMCs is a complex process. The results provide new insights on MMCs activation.

Altered gene expression signatures by calcitonin gene-related peptide promoted mast cell activity in the colon of stress-induced visceral hyperalgesia mice.

Calcitonin gene-related peptide (CGRP) is possibly involved in recruitment of mucosal mast cells (MCs) in the gut that may be associated with the development of irritable bowel syndrome (IBS), but the role of CGRP on the activation of MCs is still unknown. Using RNA sequencing (RNA-seq), we examined differentially expressed genes (DEGs) in mouse MCs following CGRP treatment. The expression of key genes in colonic MCs and their relationship with CGRP-containing fibers were examined by immunofluorescence in chronic water-avoidance stress (WAS)-induced visceral hyperalgesia mice. A total of 29 DEGs were found significantly changed with 28 upregulated and 1 downregulated following treatment of MCs with CGRP. Bioinformatics analysis showed that key higher DEGs included those associated with response to corticotropin-releasing hormone (CRH), regulation of transcription, MC activation, and proliferation. These processes are enriched for genes associated with stress-induced MC activation in IBS. Western blot verified changes in representative DEGs (Nr4a3, Crem, Gpr35, FosB, Sphlk1) and real-time cell analysis (RTCA) verified the MC proliferation. The vast majority of colonic MCs nearly CGRP-containing fibers in WAS mice overexpressed only Nr4a3 with little to no FosB, Gpr35, Sphlk1, or Crem expression. Nr4a3 knockdown may attenuate the promotion effect of CGRP on MC viability. Our results suggest that CGRP is a critical regulator of key expressed genes in MC activation. Nr4a3 as a novel regulator of MC function may have an effect on stress-induced visceral hyperalgesia, and this may represent the novel target for drug development.

Proton Pump Inhibitors Reduce Duodenal Eosinophilia, Mast Cells, and Permeability in Patients With Functional Dyspepsia

Despite the growing recognition of duodenal alterations in the pathophysiology of functional dyspepsia (FD), the effect and mechanism of proton pump inhibitors (PPIs) or first-line therapy remain unclear. We studied duodenal and systemic alterations in relation to PPI therapy in patients with FD and healthy volunteers (HVs). We performed a prospective interventional study assessing symptoms (Patient Assessment of Gastrointestinal Symptom Severity Index), duodenal alterations, and systemic factors in patients with FD ("FD-starters") and HVs before and after PPI therapy (pantoprazole 40 mg once daily for 4 weeks). Duodenal mucosal eosinophils, mast cells and permeability were quantified. Luminal pH and bile salts were determined in duodenal aspirates. Procedures were also performed in PPI-refractory patients with FD ("FD-stoppers") before and 8 weeks after PPI withdrawal. Between- and within-group changes from baseline and associations with duodenal or systemic factors were analyzed using linear mixed models. The study was completed by 30 HV, 27 FD-starters, and 18 FD-stoppers. Symptoms and duodenal eosinophils, mast cells (all, P < .0001), and paracellular passage (P= .02) were significantly higher in FD-starters vs HVs and reduced with PPI therapy. Symptoms and duodenal immune cells also decreased in FD-stoppers off PPIs. In contrast, immune cells and permeability increased in HVs on PPIs. Dyspeptic symptoms correlated with eosinophils before and during PPI therapy, and increased eosinophils and permeability in HVs on PPIs were associated with changes in bile salts. We provide the first prospective evidence for eosinophil-reducing effects as a therapeutic mechanism of PPIs in FD, with differential effects in HVs pointing to a role of luminal changes. ClinicalTrials.gov, Number: NCT03545243.

Extended Cleavage Specificity of the Rat Vascular Chymase, a Potential Blood Pressure Regulating Enzyme Expressed by Rat Vascular Smooth Muscle Cells

Serine proteases constitute the major protein content of the cytoplasmic granules of several hematopoietic cell lineages. These proteases are encoded from four different loci in mammals. One of these loci, the chymase locus, has in rats experienced a massive expansion in the number of functional genes. The human chymase locus encodes 4 proteases, whereas the corresponding locus in rats contains 28 such genes. One of these new genes has changed tissue specificity and has been found to be expressed primarily in vascular smooth muscle cells, and therefore been named rat vascular chymase (RVC). This β-chymase has been claimed to be a potent angiotensin-converting enzyme by cleaving angiotensin (Ang) I into Ang II and thereby having the potential to regulate blood pressure. To further characterize this enzyme, we have used substrate phage display and a panel of recombinant substrates to obtain a detailed quantitative view of its extended cleavage specificity. RVC was found to show a strong preference for Phe and Tyr in the P1 position, but also to accept Leu and Trp in this position. A strong preference for Ser or Arg in the P1’ position, just C-terminally of the cleavage site, and a preference for aliphatic amino acids in most other positions surrounding the cleavage site was also seen. Interesting also was a relatively strict preference for Gly in positions P3’ and P4’. RVC thereby shares similarity in its specificity to the mouse mucosal mast cell chymase mMCP-1, which efficiently converts Ang I to Ang II. This similarity adds support for the role of β-chymases as potent angiotensin converters in rodents, as their α-chymases, which have the capacity to efficiently convert Ang I into Ang II in other mammalian lineages, have become elastases. However, interestingly we found that RVC cleaved both after Arg2 and Phe8 in Ang I. Furthermore this cleavage was more than two hundred times less efficient than the consensus site obtained from the phage display analysis, indicating that RVC has a very low ability to cleave Ang I, raising serious doubts about its role in Ang I conversion.

IL-4–BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. Abone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an invitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

Protective effect of bergapten in acetic acid-induced colitis in rats

Bergapten (5-methoxysporalen) is a furanocoumarin extracted from several species of citrus and bergamot oil. Bergamot essential oil is used traditionally in the management of inflammatory conditions. Previous studies on bergapten have explored mainly its in vitro anti-inflammatory activities which include suppression of the expression and release of pro-inflammatory cytokines such as TNF-α and interleukins as well as prostaglandins. Bergapten enhances the clearance of neutrophils and macrophages from the site of inflammation and reduces oxidative stress by inhibition of reactive oxygen species (ROS). Bergapten was assessed for its anti-inflammatory properties in acetic acid-induced colitis. Animals were obtained and randomly placed in six (6) groups (n = 5) after acclimatization. Colitis was induced by rectal administration using 4% v/v acetic acid in Sprague Dawley rats after pre-treatment for 5 days. Bergapten was administered at doses of 3, 10, and 30 mg kg−1p.o. while the control group received saline 5 mL kg−1p.o. and the standard drug employed was sulphasalazine at a dose of 500 mg kg−1. Assessments made for colon-weight-to-length ratio, colonic injury, and mucosal mast cell degranulation. There were reduced colon-weight-to-length ratios in animals treated with bergapten which was significant (p < 0.5) for doses 10 and 30 mg kg−1 compared to the disease control group Both macroscopic and microscopic damage were reduced as well, with a lesser percentage of degranulated mast cells. Macroscopic damage was reduced for bergapten at doses 10 and 30 mg kg−1 significantly at p < 0.5 and p < 0.001, respectively. Similarly, microscopic damage was reduced at p < 0.01 and p < 0.001 respectively for bergapten 10 and 30 mg kg−1. The reduction of degranulation by bergapten was significant at p < 0.001. There was generally reduced damage at inflammatory sites as well as decreased infiltration of inflammatory cells. Overall, bergapten reduces inflammation in acetic acid-induced colitis.

Limited role of mast cells during infection with the parasitic nematode Litomosoides sigmodontis.

Mast cells are innate effector cells that due to their localization in the tissue form the first line of defense against parasites. We have previously shown that specifically mucosal mast cells were essential for the termination of the intestinal Strongyloides ratti infection. Here, we analyze the impact of mast cells on the immune response and defense against the tissue-dwelling filarial nematode Litomosoides sigmodontis using mast cell-deficient Cpa3cre mice. Despite an increase and an activation of mast cells at the site of infection in wildtype BALB/c mice the outcome of L. sigmodontis infection was not changed in mast cell-deficient BALB/c Cpa3cre mice. In Cpa3cre mice neither vascular permeability induced by blood-sucking mites nor the migration of L3 was altered compared to Cpa3 wildtype littermates. Worm burden in the thoracic cavity was alike in the presence and absence of mast cells during the entire course of infection. Although microfilaremiae in the peripheral blood increased in mast cell-deficient mice at some time points, the infection was cleared with comparable kinetics in the presence and absence of mast cells. Moreover, mast cell deficiency had no impact on the cytokine and antibody response to L. sigmodontis. In summary, our findings suggest that mast cells are not mandatory for the initiation of an appropriate immune response and host defense during L. sigmodontis infection in mice.