Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice

Abstract

Background: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment.

Methods: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain.

Results: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1β induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1β and TNFα augmented the barrier injury.

Conclusions: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.

Keywords: Blood–brain barrier; Lung infection; Permeability.

Fig. 1

Effects of PA pneumonia on lung inflammation. a Schematic diagram showing experimental design. Animals were exposed to PA on day 0 and then experiments were performed 24 h, 7-days or 1-month post-infection. b Representative NIR fluorescence images of the left lung lobe obtained from control and PA infected mice at 24 h post-infection (OD₆₀₀ 0.3) showing the distribution of 70-kDa tracer within the lung tissue. c Summary data showing lung permeability of 70-kDa tracer. Mann–Whitney test, P = 0.016 vs. control (uninfected) group. d Plasma leakage indicated by increased levels of albumin in bronchoalveolar lavage fluid (BALF) obtained from control and PA-infected mice at different time points post-infection (6 h, 24 h, and 7 days). Kruskal–Wallis test, P = 0.0001, P = 0.001 vs. control (uninfected) group. e Histological analysis by conventional H&E staining of lung sections in control and PA-infected mice 24 h post-infection (OD₆₀₀ 0.3). f Representative confocal images of neutrophil immunodetection (red), lectin-positive brain endothelial cells (green) and DAPI (nuclei; blue) in the brain from a control (up) and a PA-lung infected mouse 24 h post-infection (bottom). In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M

Fig. 2

Effects of PA lung infection on BBB permeability and cell–cell junction expression. Representative NIR fluorescence images of whole brains obtained from control and PA infected mice (OD₆₀₀ 0.3) showing the distribution of (a) 10-kDa and (c) 70-kDa tracers within the tissue. b, d Summary data showing BBB paracellular permeability of 10-kDa and 70-kDa fluorescence tracers. Mann–Whitney test, P < 0.05 vs. control (uninfected) group. e Time course of BBB permeability changes to small size solutes as measured by NaFl uptake starting at 24 h post-infection to 1-month post-infection (OD₆₀₀ 0.3). Kruskal–Wallis test, P = 0.01 and P = 0.0002 vs. control (uninfected) group. f Summary data showing mRNA expression levels of VE-cadherin, occludin and claudin-5 in brains harvested from control and PA infected mice at 24 h, 7 days and 1 month after infection. Kruskal–Wallis test, P = 0.01 and P = 0.003 and P = 0.002 vs. control (uninfected) group. g VE-cadherin, occludin and claudin-5 immunoblotting in brain homogenates obtained from control and lung infected (24 h) mice, and quantification of their protein abundance relative to β-actin loading control. h Representative confocal images of brain cortex from control and PA-infected (24 h) mice of VE-cadherin and claudin-5 (red) co-stained with lectin (blue, magenta-merged). In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M

Fig. 3

Effect of PA pneumonia on mouse behavior. a Representative tracks of control and PA-infected mice (OD₆₀₀ 0.3) recorded by ANYMaze^® video system during the open field test. b Total distance traveled by control and PA-infected mice. c Time mice remained immobile in the open field. d Time spent in the center of the open field by controls and PA-infected mice. Mann–Whitney test, P = 0.004, P = 0.02, P = 0.01 vs. control (uninfected) group. In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M. In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M

Fig. 4

PA load in peripheral organs and brain regions. a Bacterial growth in lung and spleen (CFUs per mg of tissue) and brain (in CSF; CFU per μL) obtained from infected animals (OD₆₀₀ 0.3) and controls after intratracheal administration of PA at 24 h, 7-days and 1-month post-infection. b Representative confocal micrograph showing (b) lung and (c) spleen sections obtained from GFP-labelled PA infected animals at 24 h post-infection (OD₆₀₀ 0.3) and controls. PA is labelled in green and nuclei (DAPI) in blue. d–g Representative confocal micrographs of (d) cortical sections, (e) choroid plexus (f) hippocampus and (g) isolated capillaries showing the lack of distribution of PA (GFP-labelled PA) in the brain of infected mice at 24 h post-infection and controls. Lectin (red) was used to label the blood vessels in tissue sections and claudin-5 or ZO-1 (red) to label isolated capillaries. DAPI (blue) was used to stain the nuclei

Fig. 5

Appearance of leukocytes in CNS during PA-induced lung infection. a White blood counts in control and PA-infected animals (OD₆₀₀ 0.3). Mann–Whitney test, P = 0.016 vs. control (uninfected) group. b Neutrophil and monocyte counts in blood obtained from control and PA-infected mice (OD₆₀₀ 0.3) measured by ProCyte One hematology analyzer. Mann–Whitney test, P = 0.04, P = 0.03 vs. control (uninfected) group. c Flow cytometry analysis of brain tissue homogenates labeled with anti-CD45 and anti-CD11b antibodies with gating on CD45⁺ cells. Quantitative analyses of (d) infiltrated leukocytes and (e) microglia in control and PA infected mice. Mann–Whitney test, P = 0.03 vs. control (uninfected) group. In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M. f Representative confocal images of infiltrated leukocytes (CD45⁺; green) in the brain from a control (left) and a PA-lung infected mouse (right). DAPI (nuclei; blue). g GFAP immunoblotting in brain homogenates from control and PA-infected (24 h after infection) mice, and quantification of GFAP protein abundance relative to β-actin loading control. Mann–Whitney test, P = 0.016 vs. control (uninfected) group. h Representative confocal images of GFAP immunodetection (red), lectin-positive brain endothelial cells (green) and DAPI (nuclei; blue) in the brain of a control (left) and a PA-lung infected mouse (right). The inset in each main image (control and infected) represents a closer view of the indicated region at a higher magnification (right panels). In all graphs, each point indicates data from an individual animal, bar graphs (columns and error bars) show mean ± S.E.M

Fig. 6

PA lung infection causes systemic and neuroinflammation. a–c PA-induced plasma levels of IL-1β, IL-6 and TNF-α at different time points after infection (OD₆₀₀ 0.3) compared to uninfected controls. Kruskal–Wallis test, P = 0.01, P = 0.001 and P = 0.0009 vs. control (uninfected) group. d–f mRNA expression levels of (d) cytokines (IL-1β, IL-6 and TNF-α), (e) chemokines (CXCL1 and CXCL2) and (f) adhesion molecules (ICAM-1 and VCAM-1) in cortex from control and infected. Mann–Whitney test, P = 0.004, P = 0.03, P = 0.001, P = 0.001, P = 0.02 vs. control (uninfected) group. g–i mRNA expression levels of (g) cytokines (IL-1β, IL-6 and TNF-α), (h) chemokines (CXCL1 and CXCL2) and (i) adhesion molecules (ICAM-1 and VCAM-1) in hippocampus from control and infected mice. Mann–Whitney test, P = 0.01, P = 0.005 vs. control (uninfected) group. In all graphs, each point indicates data from an individual animal bar graphs (columns and error bars) show mean ± S.E.M

Fig. 7

Effect of proinflammatory cytokines on brain microvascular endothelial barrier function. a TER measurements across confluent monolayers of mouse brain microvascular endothelial cells treated with IL-1β (2–200 ng/mL) and IL-1β + TNF-α (20 ng/mL each). The data were represented as resistance change. Mean ± S.E.M., n = 3. Arrow indicates when cytokines were added. b Summary data showing TER changes in brain microvascular endothelial cells treated with IL-1β and IL-1β + TNF-α. Kruskal–Wallis test with Dunn’s multiple comparisons test, P < 0.0001, IL-1β (2–200 ng/mL) vs. control (vehicle) group; P < 0.0001, IL-1β + TNF-α (20 ng/mL each) vs. control (vehicle). c Immunocytochemical analysis of claudin-5, VE-cadherin and ZO-1 (red) on brain microvascular endothelial cells under control conditions (vehicle) and after IL-1β (20 ng/mL) treatment in vitro. Nuclei was stained with DAPI. White arrows indicate TJ disorganization