Abstract
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment. To date, in vivo functional approaches have been limited by the inaccessibility to the alveolar sacs, which are located at the anatomical terminal of the respiratory duct in mammals. We are the first to characterize the swim bladder of the zebrafish larva, which is similar to the mammalian lung, as a real-time in vivo model for examining pulmonary neutrophil infiltration during ALI. We observed that the delivery of exogenous materials, including lipopolysaccharide (LPS), Poly IC and silica nanoparticles, by microinjection triggered significant time- and dose-dependent neutrophil recruitment into the swim bladder. Neutrophils infiltrated the LPS-injected swim bladder through the blood capillaries around the pneumatic duct or a site near the pronephric duct. An increase in the post-LPS inflammatory cytokine mRNA levels coincided with the in vivo neutrophil aggregation in the swim bladder. Microscopic examinations of the LPS-injected swim bladders further revealed in situ injuries, including epithelial distortion, endoplasmic reticulum swelling and mitochondrial injuries. Inhibitor screening assays with this model showed a reduction in neutrophil migration into the LPS-injected swim bladder in response to Shp2 inhibition. Moreover, the pharmacological suppression and targeted disruption of Shp2 in myeloid cells alleviated pulmonary inflammation in the LPS-induced ALI mouse model. Additionally, we used this model to assess pneumonia-induced neutrophil recruitment by microinjecting bronchoalveolar lavage fluid from patients into swim bladders; this injection enhanced neutrophil aggregation relative to the control. In conclusion, our findings highlight the swim bladder as a promising and powerful model for mechanistic and drug screening studies of alveolar injuries.
Highlights
Acute lung injury (ALI) and its devastating clinical syndrome, acute respiratory distress syndrome (ARDS), are characterized by rapid respiratory failure, refractory arterial hypoxemia, pulmonary edema and bilateral infiltrates accompanied by pleural effusions.[1]
Zebrafish models have been utilized to investigate the pathogeneses of lung diseases, including idiopathic pulmonary fibrosis modeled by embryo xenotransplantation,[17] the host–pathogen interactions that occur after bacterial infections[18] and tailfin injury-induced neutrophilic inflammation.[19]
To model the aforementioned stimulation in zebrafish, we microinjected the exogenous materials into the swim bladders of 5-dpf larvae as previously described[35] and investigated the neutrophil response
Summary
Acute lung injury (ALI) and its devastating clinical syndrome, acute respiratory distress syndrome (ARDS), are characterized by rapid respiratory failure, refractory arterial hypoxemia, pulmonary edema and bilateral infiltrates accompanied by pleural effusions.[1]. Air-filled sacs at the terminal ends of the distal airways, have recently garnered attention as unique compartments that are vulnerable to various environmental pathogenic attacks.[4] Exogenous materials, such as bacteria, viruses and air pollutants, can enter the alveolar microenvironment and trigger ALI/ARDS.[5,6,7] In addition to their important host defense functions, neutrophils play key roles in promoting ALI/ARDS progression by infiltrating the pulmonary alveoli and releasing granule proteins or reactive oxygen species.[8] ALI is alleviated in animal models when neutrophilic chemotaxis, migration, adhesion and transcellular diapedesis are impaired.[9,10,11] Despite decades of research, the underlying mechanism behind alveolar neutrophil infiltration during ALI remains unclear This can be attributed in part to a lack of in vivo animal models for observing the dynamics of alveolar neutrophil recruitment.[12]. These studies do not provide direct and definitive evidence for the feasibility and effectiveness of zebrafish as an in vivo model for lung diseases
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