Manipulating the air-filled zebrafish swim bladder as a neutrophilic inflammation model for acute lung injury

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.

Figure 1

Neutrophils migrate into the LPS-injected zebrafish swim bladder. (a) The dose response for neutrophil recruitment into the swim bladder of 5-dpf zebrafish larvae after stimulation with LPS, Poly IC and Nano-SiO₂ for 4 hpi. (b) Neutrophil accumulation into the Tg(mpo:GFP) zebrafish (5 dpf) swim bladder after the 50 ng LPS injection was imaged by confocal microscopy at 5 hpi (maximum projection slices n=66; green, neutrophil; scale bar: 60 μm). (c) Neutrophil recruitment to the LPS-injected swim bladder (indicated by the dotted line) of Tg(mpo:GFP) zebrafish larvae at 5 dpf was monitored (green, neutrophil; scale bar: 100 μm). (d) The neutrophil distribution (green) throughout the whole body (scale bar: 100 μm). (e) HE staining of zebrafish (5 dpf) swim bladders after the LPS challenge (scale bar: 20 μm)

Figure 2

Neutrophils are recruited to the LPS-injected swim bladders in vivo. Neutrophils accumulate in the swim bladders of Tg(mpo:GFP/flk1:mCherry) zebrafish larva after the LPS (50 ng) injection (green, neutrophil; red, vessels; z-stack: 31 sections every 5 μm; scale bar: 70 μm; arrow indicates a neutrophil that is moving into the swim bladder; time is expressed as h:min:s starting at 40 min post injection; see Supplementary Movie S1). The magnification is presented in the top right corner of each image

Figure 3

LPS induces inflammatory cytokine expression and injury in the zebrafish swim bladder. (a) Whole-body mRNA levels for IL-1β, IL-6, TNF-α and TNF-β increased after the 50-ng LPS injection. The results are presented as the mean±S.E.M.; n=30 in each group; *P<0.05; **P<0.01; (Student's t-test); three independent experiments. (b) The changes to the mRNA levels for IL-1β, IL-6, TNF-α and TNF-β in the swim bladder after the 50-ng LPS injection. n=100 swim bladders per group. (c) Damaged epithelial layer in the swim bladder of LPS-injected (50 ng) zebrafish larvae at 5 dpf (indicated by black arrows). (d) Swollen ER (indicated by white arrowheads) and the control (indicated by white arrows). Mitochondrial injuries include mitochondrial cristae breakage (indicated by asterisks) and vacuolation (indicated by black arrowheads). The control mitochondria are indicated by black arrows. The lowers panels (scale bar: 0.25 μm) are magnifications of the upper panels (scale bar: 0.5 μm)

Figure 4

Inhibitors affect neutrophil recruitment into the swim bladder after LPS injection. (a) Administration of PHPS1, LY294002, PD98059 and SP600125 inhibited neutrophil recruitment after the LPS injection (dotted line indicates swim bladders; scale bar: 100 μm). (b) The corresponding statistical graph (C, control; V, vehicle). The results are presented as the mean±S.E.M.; n=15 per group; *P<0.05; ***P<0.001 (Student's t-test)

Figure 5

Shp2 inhibition relieves LPS-induced inflammation in the murine lung. (a) BALF analysis of control (Shp2^fl/fl) and PHPS1-treated mice that were challenged with saline or LPS (3 mg/kg) for 24 h. The number of total leukocytes, neutrophils and macrophages decreased in BALF after Shp2 inhibition (n=10) or knockout (n=8). The results are presented as the mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (b) The IL-1β, IL-6, and TNF-α levels in the BALF and the CCL2, IL-1β and TNF-α mRNA levels in the lung decreased in the PHPS1-treated mice relative to the controls. The results are presented as the mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001; NS, not significant (Student's t-test). Three independent experiments. (c) HE stains of lung sections 24 h after the saline or LPS challenge (upper panels, scale bar: 100 μm; lower panels, scale bar: 100 μm). White arrowheads indicate neutrophils; black arrowheads indicate macrophages

Figure 6

Shp2 deficiency relieves LPS-induced inflammation in the murine lung. (a) BALF analysis of control (Shp2^fl/fl) and Shp2^−/− mice that were challenged with saline or LPS (3 mg/kg) for 24 h. The number of total leukocytes, neutrophils and macrophages decreased in the BALF after Shp2 inhibition (n=10) or knockout (n=8). The results are presented as the mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (b) The IL-1β, IL-6 and TNF-α levels in the BALF and the CCL2, IL-1β and TNF-α mRNA levels in the lung decreased in the Shp2^−/− mice relative to the controls. The results are presented as the mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001; NS, not significant (Student's t-test). Three independent experiments. (c) HE stains of lung sections 24 h after the saline or LPS challenge (upper panels, scale bar: 100 μm; lower panels, scale bar: 100 μm)