A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection.

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Circulating mast cell progenitors increase during natural birch pollen exposure in allergic asthma patients.

BACKGROUND: Mast cells (MCs) develop from a rare population of peripheral blood circulating MC progenitors (MCps). Here, we investigated whether the frequency of circulating MCps is altered in asthma patients sensitized to birch pollen during pollen season, compared to out of season. METHODS: Asthma patients were examined during birch pollen season in late April to early June (May), and out of season in November-January. Spirometry measurements, asthma and allergy-related symptoms, asthma control questionnaire (ACQ), and asthma control test (ACT) scores were assessed at both time points. The MCp frequency was determined by flow cytometry in ficoll-separated blood samples from patients with positive birch pollen-specific IgE, and analyzed in relation to basic and disease parameters. RESULTS: The frequency of MCps per liter of blood was higher in May than in November (p = .004), particularly in women (p = .009). Patients that reported moderate to severe asthma symptoms (<.0001), nose or eye symptoms (p = .02; p = .01), or reduced asthma control (higher ACQ, p = .01) had higher MCp frequency in May than those that did not report this. These associations remained significant after adjusting for sex and BMI. The change in asthma control to a lower ACT score in May correlated with an increase in MCp frequency in May (p = .006, rho = 0.46). CONCLUSIONS: The data suggest that the frequency of MCps increases in symptomatic patients with allergic asthma. Our results unravel a link between asthma symptoms and circulating MCps, and bring new insight into the impact of natural allergen exposure on the expansion of MCs.

Elastase- and LPS-Exposed Cpa3Cre/+ and ST2-/- Mice Develop Unimpaired Obstructive Pulmonary Disease.

IL-33 and its receptor ST2, as well as mast cells and their mediators, have been implicated in the development of chronic obstructive pulmonary disease (COPD). However, whether mast cells and the ST2 receptor play a critical role in COPD pathophysiology remains unclear. Here, we performed repeated intranasal administrations of porcine pancreatic elastase and LPS for four weeks to study COPD-like disease in wildtype, ST2-deficient, and Cpa3Cre/+ mice, which lack mast cells and have a partial reduction in basophils. Alveolar enlargement and changes in spirometry-like parameters, e.g. increased dynamic compliance and decreased expiratory capacity, were evident one day after the final LPS challenge and worsened over time. The elastase/LPS model also induced mild COPD-like airway inflammation, which encompassed a transient increase in lung mast cell progenitors, but not in mature mast cells. While ST2-deficient and Cpa3Cre/+ mice developed reduced pulmonary function uninterruptedly, they had a defective inflammatory response. Importantly, both ST2-deficient and Cpa3Cre/+ mice had fewer alveolar macrophages, known effector cells in COPD. Elastase/LPS instillation in vivo also caused increased bronchiole contraction in precision cut lung slices challenged with methacholine ex vivo, which occurred in a mast cell-independent fashion. Taken together, our data suggest that the ST2 receptor and mast cells play a minor role in COPD pathophysiology by sustaining alveolar macrophages.

IgE cross-linking induces activation of human and mouse mast cell progenitors.

BACKGROUND: The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. OBJECTIVE: Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation in vivo, we hypothesized that they are activated by IgE cross-linking. METHODS: Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. CONCLUSIONS: Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

Mast cell-derived serotonin enhances methacholine-induced airway hyperresponsiveness in house dust mite-induced experimental asthma.

BACKGROUND: Airway hyperresponsiveness (AHR) is a feature of asthma in which airways are hyperreactive to stimuli causing extensive airway narrowing. Methacholine provocations assess AHR in asthma patients mainly by direct stimulation of smooth muscle cells. Using in vivo mouse models, mast cells have been implicated in AHR, but the mechanism behind has remained unknown. METHODS: Cpa3Cre/+ mice, which lack mast cells, were used to assess the role of mast cells in house dust mite (HDM)-induced experimental asthma. Effects of methacholine in presence or absence of ketanserin were assessed on lung function and in lung mast cells in vitro. Airway inflammation, mast cell accumulation and activation, smooth muscle proliferation, and HDM-induced bronchoconstriction were evaluated. RESULTS: Repeated intranasal HDM sensitization induced allergic airway inflammation associated with accumulation and activation of lung mast cells. Lack of mast cells, absence of activating Fc-receptors, or antagonizing serotonin (5-HT)2A receptors abolished HDM-induced trachea contractions. HDM-sensitized mice lacking mast cells had diminished lung-associated 5-HT levels, reduced AHR and methacholine-induced airway contraction, while blocking 5-HT2A receptors in wild types eliminated AHR, implying that mast cells contribute to AHR by releasing 5-HT. Primary mouse and human lung mast cells express muscarinic M3 receptors. Mouse lung mast cells store 5-HT intracellularly, and methacholine induces release of 5-HT from lung-derived mouse mast cells and Ca2+ flux in human LAD-2 mast cells. CONCLUSIONS: Methacholine activates mast cells to release 5-HT, which by acting on 5-HT2A receptors enhances bronchoconstriction and AHR. Thus, M3-directed asthma treatments like tiotropium may also act by targeting mast cells.

Mast Cells and Their Progenitors in Allergic Asthma.

Mast cells and their mediators have been implicated in the pathogenesis of asthma and allergy for decades. Allergic asthma is a complex chronic lung disease in which several different immune cells, genetic factors and environmental exposures influence the pathology. Mast cells are key players in the asthmatic response through secretion of a multitude of mediators with pro-inflammatory and airway-constrictive effects. Well-known mast cell mediators, such as histamine and bioactive lipids are responsible for many of the physiological effects observed in the acute phase of allergic reactions. The accumulation of mast cells at particular sites of the allergic lung is likely relevant to the asthma phenotype, severity and progression. Mast cells located in different compartments in the lung and airways have different characteristics and express different mediators. According to in vivo experiments in mice, lung mast cells develop from mast cell progenitors induced by inflammatory stimuli to migrate to the airways. Human mast cell progenitors have been identified in the blood circulation. A high frequency of circulating human mast cell progenitors may reflect ongoing pathological changes in the allergic lung. In allergic asthma, mast cells become activated mainly via IgE-mediated crosslinking of the high affinity receptor for IgE (FcεRI) with allergens. However, mast cells can also be activated by numerous other stimuli e.g. toll-like receptors and MAS-related G protein-coupled receptor X2. In this review, we summarize research with implications on the role and development of mast cells and their progenitors in allergic asthma and cover selected activation pathways and mast cell mediators that have been implicated in the pathogenesis. The review places an emphasis on describing mechanisms identified using in vivo mouse models and data obtained by analysis of clinical samples.

In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa.

Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL1357-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL1357-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL1357-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics.

Influenza Infection in Mice Induces Accumulation of Lung Mast Cells through the Recruitment and Maturation of Mast Cell Progenitors.

Mast cells (MCs) are powerful immune cells that mature in the peripheral tissues from bone marrow (BM)-derived mast cell progenitors (MCp). Accumulation of MCs in lung compartments where they are normally absent is thought to enhance symptoms in asthma. The enrichment of lung MCs is also observed in mice subjected to models of allergic airway inflammation. However, whether other types of lung inflammation trigger increased number of MCp, which give rise to MCs, is unknown. Here, mouse-adapted H1N1 influenza A was used as a model of respiratory virus infection. Intranasal administration of the virus induced expression of VCAM-1 on the lung vascular endothelium and an extensive increase in integrin ß7hi lung MCp. Experiments were performed to distinguish whether the influenza-induced increase in the number of lung MCp was triggered mainly by recruitment or in situ cell proliferation. A similar proportion of lung MCp from influenza-infected and PBS control mice were found to be in a proliferative state. Furthermore, BM chimeric mice were used in which the possibility of influenza-induced in situ cell proliferation of host MCp was prevented. Influenza infection in the chimeric mice induced a similar number of lung MCp as in normal mice. These experiments demonstrated that recruitment of MCp to the lung is the major mechanism behind the influenza-induced increase in lung MCp. Fifteen days post-infection, the influenza infection had elicited an immature MC population expressing intermediate levels of integrin ß7, which was absent in controls. At the same time point, an increased number of toluidine blue+ MCs was detected in the upper central airways. When the inflammation was resolved, the MCs that accumulated in the lung upon influenza infection were gradually lost. In summary, our study reveals that influenza infection induces a transient accumulation of lung MCs through the recruitment and maturation of MCp. We speculate that temporary augmented numbers of lung MCs are a cause behind virus-induced exacerbations of MC-related lung diseases such as asthma.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.