A multiple comorbidities mouse lung infection model in ApoE‑deficient mice

Abstract

Acute pneumonia is characterised by a period of intense inflammation. Inflammation is now considered to be a key step in atherosclerosis progression. In addition, pre-existing atherosclerotic inflammation is considered to play a role in pneumonia progression and risk. In the present study, a multiple comorbidities murine model was used to study respiratory and systemic inflammation that results from pneumonia in the setting of atherosclerosis. Firstly, a minimal infectious dose of Streptococcus pneumoniae (TIGR4 strain) to produce clinical pneumonia with a low mortality rate (20%) was established. C57Bl/6 ApoE ^-/- mice were fed a high-fat diet prior to administering intranasally 10⁵ colony forming units of TIGR4 or phosphate-buffered saline (PBS). At days 2, 7 and 28 post inoculation (PI), the lungs of mice were imaged by magnetic resonance imaging (MRI) and positron emission tomography (PET). Mice were euthanised and investigated for changes in lung morphology and changes in systemic inflammation using ELISA, Luminex assay and real-time PCR. TIGR4-inoculated mice presented with varying degrees of lung infiltrate, pleural effusion and consolidation on MRI at all time points up to 28 days PI. Moreover, PET scans identified significantly higher FDG uptake in the lungs of TIGR4-inoculated mice up to 28 days PI. The majority (90%) TIGR4-inoculated mice developed pneumococcal-specific IgG antibody response at 28 days PI. Consistent with these observations, TIGR4-inoculated mice displayed significantly increased inflammatory gene expression [interleukin (IL)-1β and IL-6] in the lungs and significantly increased levels of circulating inflammatory protein (CCL3) at 7 and 28 days PI respectively. The mouse model developed by the authors presents a discovery tool to understand the link between inflammation related to acute infection such as pneumonia and increased risk of cardiovascular disease observed in humans.

Keywords: ApoE-deficient mice; animal model; atherosclerosis; cardiovascular disease; pneumonia.

Figure 1

Body weight gain and survival rate. (A) Experimental flow chart. Mice underwent MRI at specific time points (2, 7 or 28 days PI). Blood was also collected before the switch to a HFD, 5 weeks into a HFD, pre-inoculation and at the study end-point (2, 7 or 28 days PI). (B) In parallel, a group of 19 mice were similarly treated but assigned for PET imaging. Due to the presence of radiotracer, only blood was collected prior to inoculation and imaging otherwise no tissues were harvested from these mice. (C) Weight loss of mice intranasally inoculated with 10⁵ TIGR4 or PBS. (D) Survival curve of mice intranasally inoculated with TIGR4 or PBS. MRI, magnetic resonance imaging; PI, post inoculation; HFD, high fat diet; PET, positron emission tomography; PBS, phosphate-buffered saline.

Figure 2

FDG-PET images from TIGR4 Streptococcus-infected mice. (A-C) Representative images from a TIGR4-infected mouse at 28 days PI. (D-F) Representative images from a PBS-inoculated mouse at 28 days PI. Rows A and D are PET scan, B and E are CT for localisation, and C and F are fused PET and CT scans. Regions of interest in row C demonstrated increased FDG uptake in TIGR4-inoculated mice compared to PBS inoculated mice (row F). PET, positron emission tomography; PI, post inoculation; PBS, phosphate-buffered saline; PET, positron emission tomography; CT, computed tomography; FDG, fluorodeoxyglucose.

Figure 3

Average and maximum lung standardised uptake values on PET imaging. Across all time points there was increased fluorodeoxyglucose uptake in the lung of TIGR4-inoculated mice compared to PBS-inoculated mice. (A and B) At 7-days post inoculation there was a significant increase (P=0.0159) in TIGR4-inoculated mice compared to PBS in both the average and maximum lung standardised uptake values. PET, positron emission tomography; PBS, phosphate-buffered saline; SUV, standardised uptake values.

Figure 4

MRI of the lungs. (A and D) Representative images of mouse lungs from a PBS- and TIGR4-inoculated mouse at 2 days PI. ROI in D, demonstrates left and right lung consolidation with infiltration in the TIGR4-inoculated mouse. (B and C) Representative images of lungs from a PBS- and TIGR4-inoculated mouse at 7 days PI. ROI highlighted in (E) displays infiltrate in the right middle lobe. (C and F) Representative images of lungs from a PBS- and TIGR4-inoculated mouse at 28 days PI. ROI in F, demonstrates pleural effusion in the right lung. All scans shown are T1 weighted images. MRI, magnetic resonance imaging; PBS, phosphate-buffered saline; PI, post inoculation; ROI, region of interest.

Figure 5

S. pneumoniae-specific serum antibody levels. Blood was collected from phosphate-buffered saline (control) or TIGR4-inoculated mice at specific PI time points: (A) 2 days PI, (B) 7 days PI and (C) 28 days PI. Dotted horizontal line represents average + 3x standard deviation. Solid horizontal line represents the median. PI, post inoculation; PBS, phosphate-buffered saline; OD, optical density.

Figure 6

Representative images of lung remodelling in TIGR4- and PBS-inoculated mice at 28 days post inoculation. (A) Tissue sections were stained with hematoxylin and eosin or Trichrome. Rows 1 and 2 captured at a magnification of x10, and row 3 captured at a magnification of x20. All magnification bars represent 100 µm. (B) Average lung collagen content per µm in TIGR4- and PBS-inoculated mice including and excluding bronchioles. PBS, phosphate-buffered saline; H&E, hematoxylin and eosin.

Figure 7

Gene expression of inflammatory mediators in the lungs. Phosphate-buffered saline control or TIGR4-inoculated mice were sacrificed at specific time points and RNA was extracted from lung tissues. mRNA expression levels of (A) IL-1β, (B) IL-6, (C) TGF-β, (D) TNF-α, and (E) IFN-γ were quantified by real-time polymerised chain recation PCR and normalised against two housekeeping genes (GAPDH and HPRT). The solid horizontal line represents the median and the dotted line represents no change in gene expression. RNA, ribonucleic acid; PBS, phosphate-buffered saline; IL-1β, interleukin-1β; IL-6, interleukin-6; TGF-β, tumor growth factor-β; TNF-α, tumor necrosis factor; IFN-γ, interferon-γ.

Figure 8

Circulating levels of soluble proteins. Blood was collected from phosphate-buffered saline (control) or TIGR4-inoculated mice at specific time points. Levels of circulating (A) dickkopf-1, (B) chemokine (C-C motif) ligand 3 (also known as macrophage inflammatory protein 1-α, (C) interleukin-5, (D) interleukin-1β, (E) interleukin-6, (F) interleukin-10, (G) interleukin-17, and (H) matrix metalloproteinase-12 were assessed. Horizontal line represents the median. Dotted line represents minimum level of detection of the Luminex assay. N values: 2 days post inoculation: Inf=5 and Control=3; 7 days post inoculation: Inf=12 and Control=12; 28 days post inoculation: Inf=17 and Control=11. PBS, phosphate-buffered saline.