Abstract The mast cells are an integral part of the human immune system. They are important modulators of inflammatory and physiological processes. Mast cells exert their functions through degranulation and release of inflammatory mediators, such as histamine, proteases and cytokines. There are two main pathways leading to the mast cell activation, the immunoglobulin-dependent and the immunoglobulin-independent pathway. The latter pathway can be triggered by several non-immunological stimuli, and two novel receptors responsible for the activation have been identified, the MAS-related G protein-coupled receptor X1 (MRGPRX1) and X2. The MRGPRX1 and MRGPRX2 have two established functions: i) they trigger the degranulation of mast cells and ii) they are involved in pain perception and itch on a specific subset of sensory neurons. These receptors are not expressed in all of the populations of mast cells, only in the tryptase and chymase containing mast cells, contributing to the mast cell heterogeneity. Unlike most G protein-coupled receptors, the MRGPRX1 and MRGPRX2 are quite non-selective, binding an ever growing list of different ligands. Their ligands include endogenous neuropeptides, host defense peptides and protein fragments, as well as synthetic compounds such as different antibiotics. Their endogenous ligands could be a triggering signal in some mast cell-related diseases by degranulating mast cells and thereby inducing inflammation. Due to the non-selectivity of MRGPRX1 and MRGPRX2, they probably still have many hitherto unknown ligands. The aim of this study was to isolate novel endogenous ligands for the MRGPRX1 and MRGPRX2 from human tissues with the “reverse pharmacology approach” and to determine their potential to degranulate mast cells. The starting materials for the isolation, human platelets and plasma, contained MRGPRX1 and MRGPRX2 activating compounds. From the human plasma, three fragments of albumin able to activate the MRGPRX2 were isolated and sequenced. These fragments were dose-dependently activating the MRGPRX2 and degranulating mast cells. Two MRGPRX1 activating hemoglobin β-chain fragments were isolated from human platelets. These fragments were dose-dependently activating the MRGPRX1, but had no effect on mast cell degranulation. / Tiivistelmä Syöttösolut on tärkeä osa ihmisen immuunijärjestelmää. Ne ovat tärkeitä tulehdus- ja fysiologistenprosessien säätelijöitä. Syöttösolujen vaikutus välittyy degranulaation ja siinä vapautuvien tulehdusvälittäjäaineiden kautta. Vapautuviin aineisiin lukeutuu esim. histamiini ja lukuisia sytokiinejä, sekä proteaaseja. Syöttösolujen aktivaatio voi tapahtua immunoglobuliineista riippuvaa tai immunoglobuliineista riippumatonta reittiä pitkin. Monet ei-immunologiset tekijät voivat laukaista jälkimmäisen reitin ja kaksi uutta tähän vaikuttavaa G-proteiinikytkentäistä reseptoria on löydetty, MAS-related G protein-coupled receptor X1 (MRGPRX1) ja X2. MRGPRX1:llä ja MRGPRX2:lla on kaksi tunnettua tehtävää: i) ne laukaisevat syöttösolujen degranulaation ja ii) ne osallistuvat kivun ja kutinan aistimiseen tietyissä tuntohermoissa. Näitä reseptoreita ei ilmennetä kaikissa syöttösoluissa, vaan ainoastaa tryptaasia ja kymaasia sisältävissä syöttösoluissa, ja täten osaltaan selittävät syöttösolujen monimuotoisuutta. Useimmista G-proteiinikytkentäisistä reseptoreista poiketen MRGPRX1 ja MRGPRX2 ovat laajakirjoisia, sitoen monia erilaisia ligandeja. Ligandeihin kuuluu endogeenisia neuropeptidejä, antimikrobiaalisia peptidejä ja proteiinin fragmentteja, sekä synteettisiä yhdisteitä kuten erilaisia antibiootteja. Reseptoreiden endogeeniset ligandit voivat toimia laukaisijana jossain syöttösoluihin liittyvissä sairauksissa, degranuloidessaan syöttösoluja ja aiheuttaen paikallisen tulehdustilan. Reseptoreiden laajakirjoisuudesta johtuen niillä on oletettavasti monia vielä tuntemattomia ligandeja. Tämän tutkimuksen tarkoitus oli eristää uusia endogeenisiä ligandeja MRGPRX1:lle ja MRGPRX2:lle ihmisen kudoksista ”kääteisfarmakologista lähestymistapaa” hyödyntäen ja selvittää ligandien kyky syöttösolujen degranulaatioon. Lähtömateriaalina käytetyt ihmisen verihiutaleet ja plasma sisälsivät MRGPRX1:ta ja MRGPRX2:ta aktivoivia yhdisteitä. Plasmasta eristettiin ja sekvensoitiin kolme albumiinin fragmenttia, jotka aktivoivat MRGPRX2:ta. Nämä fragmentit aktivoivat MRGPRX2:ta ja degranuloivat syöttösoluja annosriippuvaisesti. Kaksi MRGPRX1:tä aktivoivaa hemoglobiinin β-ketjun fragmenttia eristettiin ihmisen verihiutaleista. Nämä fragmentit tunnistettiin hemorfiineiksi ja ne aktivoivat MRGPRX1:tä annosriippuvaisesti, mutta eivät vaikuttaneet syöttösolujen degranulaatioon.

Tryptases are trypsin‐like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three‐dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active‐site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline‐rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin‐binding sequence, the tryptases have 10–13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn‐linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site‐directed mutagenesis.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver. / 2024-05-22

Five experiments were conducted comparing differential intestinal immune responses to two isolates of Eimeria acervulina (EA), EA1 and EA2. In three experiments, broiler chicks were divided into control (non-challenged), EA1, or EA2 challenged (14 days of age) groups. On day 6 post-challenge (PC), changes in body weight were determined, intestinal lesions were scored, and duodenal tissue was evaluated for morphometric alterations and mucosal mast cell responses. EA1 produced duodenal lesions and reduced villus height to crypt depth ratios when compared to controls; however, no differences were found in mast cell counts. EA2 produced differing results, and observed data were suggestive of an intestinal secretory response when compared to EA1 or controls. In Experiment 4, tissues were analyzed from day 2 through day 6 PC. Villus atrophy and crypt hyperplasia were heightened on day 5 PC in both challenged groups. Mast cell counts were significantly greater on days 3 and 4 PC in EA1 birds. In Experiment 5, EA2 oocysts were cleaned with 5.25% sodium hypochlorite to evaluate the possibility of a bacterial contaminant contributing to the pathogenesis of intestinal alterations. Weight gains were decreased by challenge and villus heights and crypt depths were significantly altered in challenged birds, resulting in lower villus to crypt ratios, however, there were no differences in mast cell number. These data are indicative of differential host response and immunovariability between different isolates of the same Eimeria species and are suggestive of mast cell involvement in coccidial immunity in broiler chickens. / Master of Science

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. / Master of Science

<p>Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.</p><p>Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.</p><p>When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes. </p><p>This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.</p><p>The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.</p> / Dissertation

KIT receptor (CD117) is a receptor tyrosine kinase crucial for homeostasis of mast cells (MCs) in tissues and recruitment to sites of inflammation and tumors in response to its ligand Stem cell factor (SCF). Gain of function mutations in KIT (e.g. D816V) are frequently observed in systemic mastocytosis and other cancer types. Src Homology 2 domain containing phosphatase-2 (SHP2 or PTPN11) is a protein tyrosine phosphatase that promotes cell proliferation, survival and motility in multiple pathways and cell types. To study SHP2 function in MCs, we generated novel MC-specific Shp2 knock-out (KO) mice (MC-shp2 KO). These mice had reduced numbers of MCs in skin and peritoneum, and defective contact hypersensitivity responses compared to control mice, consistent with SHP2 promoting MC homeostasis. Using an inducible SHP2 KO bone marrow-derived MC (BMMC) culture model, we found that SHP2 KO cells were prone to apoptosis and had no MC repopulating activity in vivo. Mechanistically, SHP2 enhanced ERK activation and downregulation of pro-apoptotic protein Bim. SHP2 KO BMMCs also had defects in chemotaxis towards SCF, due to impaired activation of a Lyn/Vav/Rac pathway in SHP2 KO BMMCs. This correlated with defects in cell spreading, and F-actin polymerization in response to SCF. Treatment of BMMCs with a SHP2 inhibitor (II-B08) also led to reduced chemotaxis, consistent with SHP2 phosphatase activity being required for KIT-induced chemotaxis. Lastly, we tested whether SHP2 regulates oncogenic KIT signaling using a P815 mouse mastocytoma model. Stable silencing of SHP2 in P815 cells led to reduced cell growth and survival in vitro, and less aggressive systemic mastocytosis development in syngeneic mice. Overall, these studies identify SHP2 as a key node in SCF/KIT and oncogenic KIT pathways, and as a potential therapeutic target in several human diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-06-25 12:03:57.818

Dexamethasone has been shown to inhibit IgE-mediated mast cell activation, and the present research investigated its role in suppressing IL-33-mediated mast cell activation. We have found that micromolar concentrations of Dexamethasone are capable of suppressing IL-33-mediated mast cell cytokine production, on several genetic backgrounds, and in not only bone marrow derived mast cells, but also peritoneal mast cells. Intracellular staining demonstrated that Dexamethasone significantly reduces expression of the IL-33 receptor, T1/ST2, in mast cells; however, the cytokine suppression is independent of T1/ST2 downregulation. At the same time, Dexamethasone pretreatment significantly reduced ERK phosphorylation, but our data suggests that inhibition occurs even prior to ERK blockade. Finally, Dexamethasone treatment in vivo reduced IL-33-mediated cytokine production and neutrophil infiltration in the murine peritoneum. Thus, Dexamethasone, a well-established therapy for inflammatory disease, can suppress IL-33-mediated mast cell activation, and may therefore be effective for treating diseases now being attributed to IL-33 effects.

Background: Mast cells are known for their role in allergy, asthma, and systemic anaphylaxis, and have been shown to play a role in inflammatory disease. Interleukin-10 can regulate inflammatory responses both in vitro and in vivo, and may be a natural regulator of mast cell activation.Objective: To examine Interleukin-10 mediated regulation of FcεRI expression and related downstream signaling molecules, and to determine how this affects mast cell function in vitro and in vivo.Methods: Mast cell FcεRI expression was evaluated with and without IL-10 treatment in human lung and skin mast cells, and on peritoneal mast cells from mice overexpressing IL-10 via injection or a transgenic model. Mast cell function was evaluated by observing responses of IL-10 treated mice to passive systemic anaphylaxis.Results: Interleukin-10 inhibited FcεRI expression on mouse and human mast cells, both in vitro and in vivo. IL-10 also suppressed expression of the key signaling molecules Syk, Fyn, Akt and Stat5. Mice chronically overexpressing IL-10 had a reduced response to passive systemic anaphylaxis, indicating impaired mast cell activation.Conclusion: Interleukin-10 suppresses mast cell FcεRI expression in vitro and in vivo, and reduces IgE-mediated activation. The anti-inflammatory effects of IL-10 may relate to its suppression of critical signaling molecules.Clinical Implications: Interleukin-10 polymorphism is associated with increased IgE levels and incidence of atopic disease; hence IL-10 dysregulation may affect atopic etiology. Further, IL-10 therapy is a possible treatment for atopic allergy and asthma.