Virtual screening for inhibitors of the human TSLP:TSLPR interaction

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Ra), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP: TSLPR complex to investigate the drugability of this complex. Two commercially available fragment libraries were screened computationally for possible inhibitors and a selection of fragments was subsequently tested in vitro. The screening setup consisted of two orthogonal assays measuring TSLP binding to TSLPR: a BLI-based assay and a biochemical assay based on a TSLP: alkaline phosphatase fusion protein. Four fragments pertaining to diverse chemical classes were identified to reduce TSLP: TSLPR complex formation to less than 75% in millimolar concentrations. We have used unbiased molecular dynamics simulations to develop a Markov state model that characterized the binding pathway of the most interesting compound. This work provides a proof-ofprinciple for use of fragments in the inhibition of TSLP: TSLPR complexation

Pentraxin 3 (PTX3) Expression in Allergic Asthmatic Airways: Role in Airway Smooth Muscle Migration and Chemokine Production

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using (3)H-thymidine incorporation, cell count and Boyden chamber assays.PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity.Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma

Directional Secretory Response of Double Stranded RNA-Induced Thymic Stromal Lymphopoetin (TSLP) and CCL11/Eotaxin-1 in Human Asthmatic Airways

Background Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Methods Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Results Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. Conclusions There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations

Investigating NF-kappa B signaling in lung fibroblasts in 2D and 3D culture systems

BACKGROUND: Inflammatory respiratory diseases are amongst major global health challenges. Lung fibroblasts have been shown to play a key role in lung inflammatory responses. However, their exact role in initiation and maintenance of lung diseases has remained elusive partly due to the limited availability of physiologically relevant in vitro models. Therefore, developing new tools that enable investigating the molecular pathways (e.g. nuclear factor-kappa B (NF-κB) activation) that underpin inflammatory responses in fibroblasts could be a valuable resource for scientists working in this area of research. RESULTS: In order to investigate NF-κB activation in response to pro-inflammatory stimuli in real-time, we first developed two detection systems based on nuclear localization of NF-κB by immunostaining and luciferase reporter assay system. Furthermore using electrospun porous scaffolds, with similar geometry to human lung extracellular matrix, we developed 3D cultures of lung fibroblasts allowing comparing NF-κB activation in response to pro-inflammatory stimuli (i.e. TNF-α) in 2D and 3D. Our data clearly show that the magnitude of NF-κB activation in 2D cultures is substantially higher than 3D cultures. However, unlike 2D cultures, cells in the 3D model remained responsive to TNF-α at higher concentrations. The more subdued and wider dynamic range of NF-κB responses in 3D culture system was associated with a different expression pattern for TNF receptor I in 3D versus 2D cultures collectively reflecting a more in vivo like TNF receptor I expression and NF-κB activation pattern in the 3D system. CONCLUSION: Our data suggest that lung fibroblasts are actively involved in the pathogenesis of lung inflammation by activation of NF-κB signaling pathway. The 3D culture detection system provides a sensitive and biologically relevant tool for investigating different pro-inflammatory events involving lung fibroblasts

Pro-inflammatory and immunomodulatory functions of airway smooth muscle: Emerging concepts

Airway smooth muscle (ASM) is the main regulator of bronchomotor tone. Extensive studies show that in addition to their physical property, human airway smooth muscle (ASM) cells can participate in inflammatory processes modulating the initiation, perpetuation, amplification, and perhaps resolution of airway inflammation. Upon stimulation or interaction with immune cells, ASM cells produce and secrete a variety of inflammatory cytokines and chemokines, cell adhesion molecules, and extracellular matrix (ECM) proteins. These released mediators can, in turn, contribute to the inflammatory state, airway hyperresponsiveness, and airway remodeling present in asthma. As our knowledge of ASM myocyte biology improves, novel bioactive factors are emerging as potentially important regulators of inflammation. This review provides an overview of our understanding of some of these molecules, identifies rising questions, and proposes future studies to better define their role in ASM cell modulation of inflammation and immunity in the lung and respiratory diseases. © 2012