IL-10 Modulates the Expression and Activation of Pattern Recognition Receptors in Mast Cells.

Mast cells (MCs) are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, and cancer, among others. MCs identify microorganisms by pattern recognition receptors (PRRs), activating a secretory response. Interleukin (IL)-10 has been described as an important modulator of MC responses; however, its role in PRR-mediated activation of MC is not fully understood. We analyzed the activation of TLR2, TLR4, TLR7 and Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mucosal-like MCs (MLMCs) and peritoneum-derived cultured MCs (PCMCs) from IL-10-/- and wild-type (WT) mice. IL-10-/- mice showed a reduced expression of TLR4 and NOD2 at week 6 and TLR7 at week 20 in MLMC. In MLMC and PCMC, TLR2 activation induced a reduced secretion of IL-6 and TNFα in IL-10-/- MCs. TLR4- and TLR7-mediated secretion of IL-6 and TNFα was not detected in PCMCs. Finally, no cytokine release was induced by NOD2 ligand, and responses to TLR2 and TLR4 were lower in MCs at 20 weeks. These findings indicate that PRR activation in MCs depends on the phenotype, ligand, age, and IL-10.

Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs.

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

In vitro and in vivo preclinical profile of abediterol (LAS100977), an inhaled long-acting ß2-adrenoceptor agonist, compared with indacaterol, olodaterol and vilanterol.

Abediterol is a novel long-acting ß2-adrenoceptor agonist (LABA) currently in development for once-daily combination maintenance therapy of asthma and COPD. This study investigated the preclinical profile of abediterol in terms of affinity, potency, selectivity, duration of action and cardiac effects in comparison to the marketed once-daily LABAs indacaterol, olodaterol and vilanterol. Abediterol was the compound with the highest in vitro potency for dog, guinea pig and human ß2-adrenoceptors. In electrical field stimulated guinea pig trachea, abediterol demonstrated 5-, 44- and 77-fold greater potency than olodaterol, indacaterol and vilanterol, respectively. In anaesthetised guinea pigs, inhaled abediterol was also the most potent compound, with 5-20 times higher bronchoprotective potency than other once-daily LABAs against acetylcholine. The bronchoprotective half-life of abediterol in guinea pigs was 36h compared with 51h for indacaterol, 47h for olodaterol, and 18h for vilanterol. In anaesthetised dogs, abediterol also inhibited acetylcholine-induced bronchoconstriction, with higher potency than olodaterol and vilanterol [ID40 (dose inhibiting bronchoconstriction by 40%) of 0.059µg/kg, 0.180µg/kg and 2.870µg/kg, respectively]. In parallel, effects on heart rate in dogs were also measured. Abediterol showed greater safety index (defined as the ratio of the maximal dose without effect on heart rate and the ID40) than olodaterol and vilanterol (10.5 versus 4.9 and 2.4, respectively). Taken together, these data suggest that abediterol offers potent bronchodilation and a sustained duration of action suited to once-daily dosing, plus a reduced potential for class-related cardiac side effects.

Enhanced cough reflex in a model of bleomycin-induced lung fibrosis in guinea pigs.

Fibrotic lung diseases, such as idiopathic pulmonary fibrosis, are associated with spontaneous dry cough and hypersensitivity to tussive agents. Understanding the pathophysiology driving enhanced cough may help us to define better therapies for patients. We hypothesized that lung fibrosis induced by intratracheal bleomycin would exacerbate the cough reflex induced by tussive agents in guinea pigs. Disease progression in the lungs was characterized at days 1, 7, 14, 21 and 28 after bleomycin administration. Inflammatory and fibrotic markers, as well as neurotrophin levels, were assessed in bronchoalveolar lavage fluid and/or lung tissue. Cough sensitivity to citric acid, capsaicin and allylisothiocyanate was evaluated in conscious animals at days 14 and 21 after bleomycin administration. Pulmonary lesions evolved from an early inflammatory phase (from day 1 to day 7) to a fibrotic stage (between days 14 and 28). Fibrosis was related to increased levels of matrix metalloproteinase-2 in bronchoalveolar lavage fluid (day 21: saline, 0.26 ng/ml; bleomycin, 0.49 ng/ml). At day 14, we also observed increased cough reflexes to citric acid (163%), capsaicin (125%) and allylisothiocyanate (178%). Cough exacerbation persisted, but at a lower extent, by day 21 for capsaicin (100%) and allylisothiocyanate (54%). Moreover, bronchoalveolar lavage fluid concentrations of brain-derived neurotrophic factor, suggested to induce nerve remodelling in chronic cough, were also enhanced (day 1: saline, 14.21 pg/ml; bleomycin, 30.09 pg/ml). In summary, our model of bleomycin-induced cough exacerbation may be a valuable tool to investigate cough hypersensitivity and develop antitussive therapies for fibrotic lung diseases.

Changes in Epithelial Barrier Function in Response to Parasitic Infection: Implications for IBD Pathogenesis.

BACKGROUND AND AIMS: Mast cells [MCs] are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we describe mast cell proteinases [chymases and tryptases] and tight junction [TJ] proteins kinetics in a rat model of postinfectious gut dysfunction. METHODS: Jejunal tissues of control and -infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and reverse transcription-quantitative polymerase chain reaction. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo. RESULTS: After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between Days 6 and 14. Thereafter, a mucosal MC hyperplasia and a late increase in connective tissue MC counts were observed. From Day 2 post-infection, TJ proteins occludin and claudin-3 expression was down-regulated whereas the pore-forming protein claudin-2 was overexpressed. The expression of proglucagon, precursor of the barrier-enhancing factor glucagon-like peptide-2, was reduced. These changes were associated with an increase in epithelial permeability, both in vitro and in vivo. CONCLUSIONS: Proteinases expression and location of mucosal and connective tissue MCs indicate a time-related pattern in the maturation of intestinal MCs following infection. Altered expression of TJ-related proteins is consistent with a loss of epithelial tightness, and provides a molecular mechanism for the enhanced epithelial permeability observed in inflammatory conditions of the gut.

IL-33-induced alterations in murine intestinal function and cytokine responses are MyD88, STAT6, and IL-13 dependent.

IL-33 is a recently identified cytokine member of the IL-1 family. The biological activities of IL-33 are associated with promotion of Th2 and inhibition of Th1/Th17 immune responses. Exogenous IL-33 induces a typical "type 2" immune response in the gastrointestinal tract, yet the underlying mechanisms remain to be fully elucidated. In addition, the role of IL-33 in the regulation of gastrointestinal function is not known. The present study investigated IL-33-dependent intestinal immunity and function in mice. Exogenous IL-33 induced a polarized type 2 cytokine response in the intestine that was entirely MyD88 dependent but STAT6 and IL-13 independent. Mice injected with recombinant IL-33 exhibited intestinal smooth muscle hypercontractility, decreased epithelial responses to acetylcholine and glucose, and increased mucosal permeability. IL-33 effects on intestinal epithelial function were STAT6 dependent, and both IL-4 and IL-13 appeared to play a role. The effects on smooth muscle function, however, were attributable to both STAT6-dependent and -independent mechanisms. In addition, IL-13 induction of insulin-like growth factor-1 was implicated in IL-33-induced smooth muscle hypertrophy. Finally, alternative activation of macrophages induced by IL-33 revealed a novel pathway that is IL-4, IL-13, and STAT6 independent. Thus manipulating IL-33 or related signaling pathways represents a potential therapeutic strategy for treating inflammatory diseases associated with dysregulated intestinal function.

PAR-2-mediated control of barrier function and motility differs between early and late phases of postinfectious gut dysfunction in the rat.

Proteinase-activated receptor-2 (PAR-2) and mast cell (MC) mediators contribute to inflammatory and functional gastrointestinal disorders. We aimed to characterize jejunal PAR-2-mediated responses and the potential MC involvement in the early and late phases of a rat model of postinfectious gut dysfunction. Jejunal tissues of control and Trichinella spiralis-infected (14 and 30 days postinfection) rats, treated or not with the MC stabilizer, ketotifen, were used. Histopathology and immunostaining were used to characterize inflammation, PAR-2 expression, and mucosal and connective tissue MCs. Epithelial barrier function (hydroelectrolytic transport and permeability) and motility were assessed in vitro in basal conditions and after PAR-2 activation. Intestinal inflammation on day 14 postinfection (early phase) was significantly resolved by day 30 (late phase) although MC counts and epithelial permeability remained increased. PAR-2-mediated ion transport (Ussing chambers, in vitro) and epithelial surface PAR-2 expression were reduced in the early phase, with a trend toward normalization during the late phase. In control conditions, PAR-2 activation (organ bath) induced biphasic motor responses (relaxation followed by excitation). At 14 days postinfection, spontaneous contractility and PAR-2-mediated relaxations were enhanced; motor responses were normalized on day 30. Postinfectious changes in PAR-2 functions were not affected by ketotifen treatment. We concluded that, in the rat model of Trichinella spiralis infection, alterations of intestinal PAR-2 function and expression depend on the inflammatory phase considered. A lack of a ketotifen effect suggests no interplay between MCs and PAR-2-mediated motility and ion transport alterations. These observations question the role of MC mediators in PAR-2-modulating postinfectious gut dysfunction.