Use of a Chemical Chaperone to Attenuate Allergic Airway Inflammation

Rationale: Asthma is a chronic inflammatory disease characterized by increased airway inflammation and fibrosis. So far, endoplasmic reticulum (ER) stress has been shown to play a role in several inflammatory diseases, however, its involvement in the pathogenesis of allergic asthma has not been clearly understood. It is also known that ER stress inhibitor chemical chaperone tauroursodeoxycholic acid (TUDCA) has been shown to attenuate inflammation in obese and diabetic conditions, but its therapeutic potential in allergic asthma is currently unknown. Objective: The current study was designed to investigate the role of ER stress in house dust mite (HDM)-induced allergic asthma, and the therapeutic efficacy of TUDCA in attenuating the hallmarks of allergic asthma (airway inflammation, mucus metaplasia and fibrosis), using a mouse model of HDM-induced allergic airways disease. Methods: Inflammation was measured by inflammatory cell counts (cell differentials) and cytokine analyses (ELISA). ER stress was examined by the expression levels of ER stress markers ATF6, ERp57, GRP78, GRP94, and CHOP. Further, changes in the airways to indicate fibrosis was determined by quantifying alpha-smooth muscle actin (α-SMA) cell expression and hydroxyproline content in the lungs. HDM and TUDCA were administered using a HDM-induced allergic airways disease murine model for preventive and therapeutic regimens. Results: TUDCA administered during the HDM-challenge phase (as a prophylactic), significantly decreased inflammatory cells and cytokines, ER stress markers, peri-bronchial collagen content and α-SMA cell expression. Moreover, TUDCA administered after the HDM-challenge phase (as a therapeutic), markedly decreased HDM-induced airway inflammation, ER stress makers, but not airway remodeling. Conclusion: These results suggest that the inhibition of ER stress by a chemical chaperone, TUDCA, could be helpful in the treatment of asthmatic patients. Gurkiranjit Kaur Rattu 2 Introduction Allergic asthma is an inflammatory disorder caused by repeated exposure to allergens resulting in chronic airway inflammation, airway remodeling with the presence of increased αSMA expression and mucus metaplasia, and airways hyperresponsiveness (1, 3, 27). Environmental and genetic factors are known to play a role in disease pathogenesis (4); however, the causes of asthma are not completely understood. Allergic airway inflammation results from initial exposure (inhaled or cutaneous), thus causing sensitization to the subsequent allergen contact (5). This event is characterized by inflammatory cells and cytokines, which respond in a coordinated, albeit dysfunctional manner (17). House dust mite (HDM) is a multifaceted allergen to which 50-80% of asthmatics are allergic (6). Nelson et al., demonstrated evidence of significantly increased Immunoglobulin E levels in response to HDM exposure as compared to other allergens such as cat, white oak, Bahia grass, and short ragweed in patients with acute asthma (7). Further, house dust mite contributes to the pathogenesis of allergic asthma in patients that are genetically pre-disposed to asthma (8). HDM exposure has been demonstrated to elicit the inflammatory response, with increases in the recruitment of eosinophils, neutrophils, macrophages, and lymphocytes (16). Thus, the HDMinduced allergic airways disease model is widely used to investigate the progression and attenuation of allergic airway inflammation and fibrosis. The endoplasmic reticulum (ER) is a network of membranes in the cell with two regions— rough and smooth. The smooth ER serves as a storage for lipid storage and secretion. In contrast, the rough ER has bound ribosomes and is responsible for synthesis and folding of proteins. In addition, it plays a key role in allergic airway inflammation (9, 13) and other inflammatory disease conditions (15, 22). Protein synthesis and folding is normally regulated in the ER under physiological conditions. However, the synthesis of proteins is augmented during pathological Gurkiranjit Kaur Rattu 3 conditions, which leads to increased demand for protein folding. These events collectively create an imbalance in protein synthesis and capacity to fold, resulting in increased accumulation of misfolded/unfolded proteins in the ER lumen, the condition referred to as ER stress. Subsequently, ER stress activates the signaling pathways to reestablish the ER homeostasis, otherwise known as the Unfolded Protein Response (UPR) (10, 22, 23). The abundance of misfolded proteins is sensed by three ER transmembrane proteins: protein kinase RNA-like ER kinase (PERK), inositolrequiring protein 1 α (IRE1 α), and activating transcription factor 6 (ATF6) (11). Sustained UPR can cause CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP)-induced cell death (10), and the accumulation of collagen in the lung (12, 13). Another key Molecular structure of TUDCA. [http://pubs.rsc.org/en/content/articlehtml/2015/ra/c5ra18985c] Chemical chaperone tauroursodeoxycholic acid (TUDCA) (molecule above), the taurine conjugate form of ursodeoxycholic acid (UDCA), has been shown to inhibit ER stress (14, 20). Exogenous administration of TUDCA has also been found to alleviate ER stress in liver and adipose tissues of diabetic and obese mice (2). In addition, TUDCA has exhibited anti-apoptotic properties in decreasing the expression of ER stress-associated proteins (20). Previously, Hoffman et al., demonstrated that the targeted knockdown of ER stress transducers ATF6 and endoplasmic reticulum resident protein 57 (ERp57) attenuated ER stress-induced airway inflammation and fibrosis (13). Moreover, there are no effective treatments for chronic allergen-induced airway inflammation and remodeling. For that reason, the ER stress inhibitor chemical chaperone TUDCA Gurkiranjit Kaur Rattu 4 might be utilized as a potential therapeutic, particularly in patients with atopic asthma and abnormal lung function. The main goal of this investigation was to determine the effect of ER stress inhibitor TUDCA on airway inflammation and remodeling in a murine model of allergic airways disease. Within this context, my primary objective was to establish a suitable method to quantitate immunohistochemical (IHC) staining of α-SMA, ERp57, and mucus metaplasia, along with the biochemical evaluation of collagen in the lungs as measurements of the pathophysiology of allergic asthma. Our results demonstrate that HDM-induced ER stress is a substantial contributor to the airways inflammation and remodeling, which can be attenuated by TUDCA.