Korcan Ayata

Production of Serotonin by Tryptophan Hydroxylase 1 and Release via Platelets Contribute to Allergic Airway Inflammation

RATIONALE 5-Hydroxytryptamine (5-HT) is involved in the pathogenesis of allergic airway inflammation (AAI). It is unclear, however, how 5-HT contributes to AAI and whether this depends on tryptophan hydroxylase (TPH) 1, the critical enzyme for peripheral 5-HT synthesis. OBJECTIVES To elucidate the role of TPH1 and the peripheral source of 5-HT in asthma pathogenesis. METHODS TPH1-deficient and TPH1-inhibitor-treated animals were challenged in ovalbumin and house dust mite models of AAI. Experiments with bone marrow chimera, mast cell-deficient animals, platelets transfusion, and bone marrow dendritic cells (BMDC) driven model of AAI were performed. 5-HT levels were measured in bronchoalveolar lavage fluid or serum of animals with AAI and in human asthma. MEASUREMENTS AND MAIN RESULTS 5-HT levels are increased in bronchoalveolar lavage fluid of mice and people with asthma after allergen provocation. TPH1 deficiency and TPH1 inhibition reduced all cardinal features of AAI. Administration of exogenous 5-HT restored AAI in TPH1-deficient mice. The pivotal role of 5-HT production by structural cells was corroborated by bone marrow chimera experiments. Experiments in mast cell-deficient mice revealed that mast cells are not a source of 5-HT, whereas transfusion of platelets from wild-type and TPH1-deficient mice revealed that only platelets containing 5-HT enhanced AAI. Lack of endogenous 5-HT in vitro and in vivo was associated with an impaired Th2-priming capacity of BMDC. CONCLUSIONS In summary, TPH1 deficiency or inhibition reduces AAI. Platelet- and not mast cell-derived 5-HT is pivotal in AAI, and lack of 5-HT leads to an impaired Th2-priming capacity of BMDC. Thus, targeting TPH1 could offer novel therapeutic options for asthma.

A Novel Function for P2Y2 in Myeloid Recipient–Derived Cells during Graft-versus-Host Disease

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2−/− mice to test the role of this receptor in GvHD. P2y2−/− recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2−/− inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2+ cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD.

The purinergic receptor subtype P2Y 2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases. The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms. Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes. Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF.

Role of purinergic signaling in experimental pneumococcal meningitis

Excessive neutrophilic inflammation contributes to brain pathology and adverse outcome in pneumococcal meningitis (PM). Recently, we identified the NLRP3 inflammasome/interleukin (IL)-1β pathway as a key driver of inflammation in PM. A critical membrane receptor for NLRP3 inflammasome activation is the ATP-activated P2 purinoceptor (P2R) P2X7. Thus, we hypothesized involvement of ATP and P2Rs in PM. The functional role of ATP was investigated in a mouse meningitis model using P2R antagonists. Brain expression of P2Rs was assessed by RT-PCR. ATP levels were determined in murine CSF and cell culture experiments. Treatment with the P2R antagonists suramin or brilliant blue G did not have any impact on disease course. This lack of effect might be attributed to meningitis-associated down-regulation of brain P2R expression and/or a drop of cerebrospinal fluid (CSF) ATP, as demonstrated by RT-PCR and ATP analyses. Supplemental cell culture experiments suggest that the reduction in CSF ATP is, at least partly, due to ATP hydrolysis by ectonucleotidases of neutrophils and macrophages. In conclusion, this study suggests that ATP-P2R signaling is only of minor or even no significance in PM. This may be explained by down-regulation of P2R expression and decreased CSF ATP levels.