Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway.

BACKGROUND: Airway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma. METHODS: The datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor. RESULTS: Both bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-ß1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression. CONCLUSIONS: Taken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma.

BACKGROUND: Airway epithelial cell (AEC) necroptosis contributes to airway allergic inflammation and asthma exacerbation. Targeting the tumor necrosis factor-like ligand 1 A (TL1A)/death receptor 3 (DR3) axis has a therapeutic effect on asthmatic airway inflammation. The role of TL1A in mediating necroptosis of AECs challenged with ovalbumin (OVA) and its contribution to airway inflammation remains unclear. METHODS: We evaluated the expression of the receptor-interacting serine/threonine-protein kinase 3(RIPK3) and the mixed lineage kinase domain-like protein (MLKL) in human serum and lung, and histologically verified the level of MLKL phosphorylation in lung tissue from asthmatics and OVA-induced mice. Next, using MLKL knockout mice and the RIPK3 inhibitor GSK872, we investigated the effects of TL1A on airway inflammation and airway barrier function through the activation of necroptosis in experimental asthma. RESULTS: High expression of necroptosis marker proteins was observed in the serum of asthmatics, and necroptosis was activated in the airway epithelium of both asthmatics and OVA-induced mice. Blocking necroptosis through MLKL knockout or RIPK3 inhibition effectively attenuated parabronchial inflammation, mucus hypersecretion, and airway collagen fiber accumulation, while also suppressing type 2 inflammatory factors secretion. In addition, TL1A/ DR3 was shown to act as a death trigger for necroptosis in the absence of caspases by silencing or overexpressing TL1A in HBE cells. Furthermore, the recombinant TL1A protein was found to induce necroptosis in vivo, and knockout of MLKL partially reversed the pathological changes induced by TL1A. The necroptosis induced by TL1A disrupted the airway barrier function by decreasing the expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin, possibly through the activation of the NF-κB signaling pathway. CONCLUSIONS: TL1A-induced airway epithelial necroptosis plays a significant role in promoting airway inflammation and barrier dysfunction in asthma. Inhibition of the TL1A-induced necroptosis pathway could be a promising therapeutic strategy.

Plasma TNFRSF11B as a New Predictive Inflammatory Marker of Sepsis-ARDS with Endothelial Dysfunction.

Inflammation plays an important role in the development of sepsis-acute respiratory distress syndrome (ARDS). Olink inflammation-related biomarker panels were used to analyze the levels of 92 inflammation-related proteins in plasma with sepsis-ARDS (n = 25) and healthy subjects (n = 25). There were significant differences in 64 inflammatory factors, including TNFRSF11B in sepsis-ARDS, which was significantly higher than that in controls. Functional analysis showed that TNFRSF11B was closely focused on signal transduction, immune response, and inflammatory response. The TNFRSF11B level in sepsis-ARDS plasma, LPS-induced mice, and LPS-stimulated HUVECs significantly increased. The highest plasma concentration of TNFRSF11B in patients with sepsis-ARDS was 10-20 ng/mL, and 10 ng/mL was selected to stimulate HUVECs. Western blot results demonstrated that the levels of syndecan-1, claudin-5, VE-cadherin, occludin, aquaporin-1, and caveolin-1 in TNFRSF11B-stimulated HUVECs decreased, whereas that of connexin-43 increased in TNFRSF11B-stimulated HUVECs. To the best of the authors' knowledge, this study was the first to reveal elevated TNFRSF11B in sepsis-ARDS associated with vascular endothelial dysfunction. In summary, TNFRSF11B may be a new potential predictive and diagnostic biomarker for vascular endothelium damage in sepsis-ARDS.

TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFß1/STAT3 Action.

PURPOSE: Asthma, an airway inflammatory disease, involves multiple tumor necrosis factors (TNF). TNF ligand superfamily member 11 (TNFSF11) and its known receptor, TNF receptor superfamily 11A (TNFRSF11A), has been implicated in asthma; however, the related mechanisms remain unknown. METHODS: The serum and bronchial airway of patients with asthma and healthy subjects were examined. The air-liquid interface of primary human bronchial epithelial (HBE) cells, and Tnfsf11+/- mouse, Tnfrsf11a+/- mouse, and a humanized HSC-NOG-EXL mouse model were established. This study constructed short hairpin RNA (shRNA) of TNFSF11, TNFRSF11A, transforming growth factor ß1 (TGFß1), and transforming growth factor ß receptor type 1 (TGFßR1) using lentivirus to further examine the ability of TNFSF11 protein. RESULTS: This study was the first to uncover TNFSF11 overexpression in the airway and serum of asthmatic human subjects, and the TNFSF11 in serum was closely correlated with lung function. The TNFSF11/TNFRSF11A axis deficiency in Tnfsf11+/- or Tnfrsf11a+/- mice remarkably attenuated the house dust mite (HDM)-induced signal transducer and activator of transcription 3 (STAT3) action and remodeling protein expression. Similarly, the HDM-induced STAT3 action and remodeling protein expression in HBE cells decreased after pretreatment with TNFSF11 or TNFRSF11A shRNA. Meanwhile, the expression of the remodeling proteins induced by TNFSF11 significantly decreased after pretreatment with-stattic (inhibitor of STAT3 phosphorylation) in HBE cells. The STAT3 phosphorylation and remodeling protein expression induced by TNFSF11 obviously decreased after pretreatment with TGFß1 or TGFßR1 shRNA in HBE cells. The above results also verified that blocking TNFSF11 with denosumab alleviated airway remodeling via the TGFß1/STAT3 signaling in the humanized HSC-NOG-EXL mice with HDM-induced asthma. CONCLUSIONS: TGFß1/STAT3 action was closely correlated with TNFSF11/TNFRSF11A axis-mediated airway remodeling. This study presented a novel strategy that blocks the TNFSF11/TNFRSF11A axis to exert a protective effect against asthma.