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
Community-acquired pneumonia (CAP) is a common and potentially fatal infection of the lungs caused by bacteria or other pathogens (
The present study was conceived with the objective of investigating the longitudinal immune responses and changes in lung morphology that occur following induction of pneumonia with
Two groups of
After 8 weeks of a HFD, mice were lightly anesthetized by inhalation with 3% isofluorane (Provet NZ Pty, Ltd.) and inoculated intranasally with 105 CFU of bacteria in a total volume of 40 µl. To determine the final dose, three factors were considered: i) The dose had to be sufficient to impose weight loss while allowing the mice the opportunity to recover from the infection; ii) the survival rate had to be >80%; and iii) pneumonia had to be detectable by magnetic resonance imaging (MRI) or positron emission tomography/computed tomography (PET/CT) imaging. The final dose was within the range of CFUs from similar mouse studies (104-106 CFU) (
Blood was collected from the tail vein, serially diluted in PBS and plated on blood agar plates. To determine bacterial burden in the lungs, supernatants of homogenised lungs were serially diluted in PBS and plated on blood agar plates. Plates were incubated for 18-24 h at 37˚C with 5% CO2 and colonies were manually counted.
Mice were fasted from food for 4-6 h (water available). The animals were warmed for 30 min (at approximately 30˚C) prior to administration of 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG). Anaesthesia with 3-5% isoflurane was administered until visible loss of consciousness and administration of ~20 MBq of 18F-FDG by either intravenous (IV) or intraperitoneal (IP) injection (volume <200 µl for IV injection). After completion of 1 h uptake, the mice were sacrificed via cervical dislocation, and PET/CT scan was completed using Bioscan BioPET/CT 105 camera (Bioscan, Inc.). Mice were sacrificed prior to imaging to reduce motion artifact from mouse orientation and movements and to overall improve image quality, and semi-quantitation. 18F-FDG-PET/CT scans were analysed by Syngio.via software (VB40; Siemens Healthineers).
Upon reaching the assigned time point, mice were sequentially MRI imaged and euthanised. Mice were placed under anaesthesia with 3-5% isoflurane under warming conditions (~30˚C). Once the depth of anaesthesia was adequate, the mouse was moved to the imaging bed where the animal was imaged using the MR Solution MRI 3T scanner (MR Solutions). Based on the signal and image quality, a gadolinium-based contrast agent was administered to improve imaging quality if deemed required by the imaging technologist. Anaesthesia with isoflurane was maintained during the scan while respiration and vital signs were monitored remotely. InVivoScope software (Bioscan, Inc.) was used for image capture according to the Harry Perkins Cancer Imaging Facility licence.
Whole lungs were harvested and embedded in Tissue-Tek OCT (ProScitech) and immediately frozen to prevent tissue damage. Samples were stored at -80˚C. Subsequently, 10-µm sections were cut, air-dried and stained at room temperature unless otherwise described. Hematoxylin and eosin (H&E; cat. no. ab245880; Abcam) was completed as per the manufacturer's instructions. Briefly, sections were fixed in 100% MeOH for 3 min before staining with hematoxylin for 5 min, 2X ddH2O wash, ~12 sec in bluing reagent, 2X ddH2O wash, dipped in 100% EtOH, counterstained for 3 min in eosin and rinsed in 100% EtOH. Trichrome staining (cat. no. ab150686; Abcam) of the frozen sections was also performed according to the manufacturer's instructions with slight modification. The collagen stain, aniline blue, was replaced with 0.5% fast green in 70% EtOH (Sigma-Aldrich; Merck KGaA). This allowed for red, green and blue colour staining and subsequent colour deconvolution using ImageJ software (v1.53k; National Institutes of Health, Inc.) (
Ribonucleic acid (RNA) from lungs was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) and reverse transcribed to complementary deoxyribonucleic acid (cDNA) using the Tetro cDNA Synthesis Kit (Meridian Bioscience) according to the manufacturer's recommendations. Messenger RNA (mRNA) levels of
Blood was collected from the tail vein and centrifuged at 541 x g for 15 min at room temperature. Serum was collected and stored at -80˚C until use. Levels of the following soluble proteins: TNF-α, IFN-γ, IL-6, IL-1β, IL-5, IL-10, IL-17, CCL3, dickkopf (Dkk)-1 and matrix metalloproteinase (MMP)-12 were assayed using the multiplex Mouse Magnetic Luminex Assay (cat. no. LXSAMSM; R&D Systems, Inc.) according to the manufacturer's instructions. Quantification of proteins were determined using the Luminex 200™ System (Thermo Fisher Scientific Inc.) via xPONENT software 4.3 (Luminex, A DiaSorin Company). The levels of detection for each analyte are: CCL3, 0.45 pg/ml; Dkk-1, 31.8 pg/ml; IFN-γ, 1.85 pg/ml; IL-1β, 41.8 pg/ml; IL-5, 0.24 pg/ml; IL-6, 2.30 pg/ml; IL-10, 8.20 pg/ml; IL-17, 7.08 pg/ml; MMP-12, 0.42 pg/ml; and TNF-α, 1.47 pg/ml.
Data are expressed as the median (range). All statistical tests were performed with GraphPad Prism 7 (GraphPad Software, Inc.). The Kaplan-Meir method was used to compare survival rates. Mann-Whitney was used to compare two groups. Differences with P-values <0.05 were considered statistically significant.
To investigate the impact of the selected inoculum of
Blood and lung tissues were collected at 2, 7 and 28 days PI. No bacteria were recovered from the lungs and there was no evidence of bacteraemia in infected
Evaluation of mouse lung PET/CT scans was performed by a level 3 trained nuclear medicine specialist (SV), who was blinded to the study groups and designations. Representative PET/CT scans are presented in
Evaluation of mouse lungs was performed by a level 3 trained radiologist (TS), who was blinded to the study groups and designations. The two mice that succumbed to the TIGR4 infection were not imaged. Of the four TIGR4-inoculated mice that underwent MRI at 2 days PI, three presented radiological findings consistent with lung infection including pleural effusion, consolidation, and various degrees of lung infiltration. In total, 4/8 (50%) TIGR4-inoculated mice scanned at 7 days PI, and 6/15 (40%) TIGR4-inoculated mice scanned at 28 days PI presented with the characteristics described above. None of the PBS-inoculated mice presented with any lung radiological abnormalities that would suggest a respiratory infection. Hence, across all time points, 13 of the 27 mice inoculated with
At 2 days PI, one TIGR4-inoculated mouse produced a low level of pneumococcal-specific IgG antibody (
H&E staining revealed lung remodelling consistent with previous studies in
There were no significant differences observed in the serum levels for any of the soluble proteins at 2 days PI between TIGR4- and PBS-inoculated mice. At 7 days PI, levels of IL-6 were significantly higher in TIGR4-inoculated mice compared to mice inoculated with PBS (
To the best of our knowledge, this is the first study to establish a longitudinal model of
It has been proposed that the introduction of a respiratory infection leads to a mounting synergistic inflammatory response that could lead to adverse cardiovascular events. In CAP, exposure of alveolar epithelial cells and resident macrophages to
In agreement with these observations, TIGR4-inoculated mice displayed delayed elevated inflammatory response in the lungs (e.g., IL-6 at 28 days PI) and systemically (e.g., CCL3 at 28 days PI). Interestingly, systemic levels of IL-6 peaked at 7 days PI. IL-6 plays an important role in linking innate to an acquired immune response by promoting differentiation of naïve CD4+ T cells (
In response to an infection, IL-6 stimulates a range of signalling pathways including NF-κB, enhancing the transcription of the mRNA of inflammatory cytokines including IL-6, TNF-α, and IL-1β. TNF-α and IL-1β in turn also activate transcription factors to produce more IL-6(
A study published by Bacci
Neutrophils play a key role in response to controlling a pneumococcal pneumonia infection (
Previously associated with lung fibrosis, epithelial cell proliferation, acute lung inflammation, increased atherosclerotic apoptosis and enlarged and destabilized plaques, Dkk-1 has been identified in both a respiratory infection and atherosclerosis setting (
In terms of lung fibrosis, a HFD and deletion of the
To date, few alternative multiple comorbidities animal models of acute pneumonia infection and atherosclerosis have been described (
There are some limitations associated with the present study. Firstly, the cellular immune responses in the lungs that may have shed light on the lack of bacterial recovery from the lung, were not investigated. As a low dose of TIGR4 bacteria was administered to achieve low mortality in the animal model, it is likely that the bacteria were cleared quickly by the immune system precluding us from bacterial recovery from lungs. Indeed, the pre-existing inflammatory response activated by atherosclerosis may have helped to clear the low intranasal dose of
Overall, the TIGR4 strain, used in studies by the authors, is an invasive strain that has been shown to cause pneumonia and lethal systemic disease following intranasal challenge (
In summary, the
The authors gratefully acknowledge Mr Lincoln Codd (Charles Gairdner Hospital, Perth, Australia), Mr Brenton O'Mara (Charles Gairdner Hospital), Ms Kirsty Richardson (Harry Perkins Institute of Medical Research, Perth, Australia), Dr Liesl Celliers (Harry Perkins Institute of Medical Research), Dr Penny Maton (University of Western Australia, Perth, Australia) and Professor Roslyn J. Francis (University of Western Australia) for technical assistance. The authors acknowledge the facilities, and the scientific and technical assistance of Microscopy Australia at the Centre for Microscopy, Characterisation and Analysis, The Cancer Imaging Facility at Harry Perkins Institute of Medical Research, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments.
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
The study was initially conceived by GW, VCM and GD. The study was developed from a combined effort of BB, SL, HPL, GW, VCM and GD. All animal work and subsequent experiments were completed by BB, SL and HPL. MRI and FDG-PET imaging analysis was completed by TS, SV, GW, VCM and GD. Manuscript preparation was performed by BB. Revisions and assessment for intellectual content was completed by SL, HPL, TS, SV, GW, VCM and GD. SL, HPL, TS and SV confirm the authenticity of all the raw data. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
All animal procedures were carried out in accordance with the Western Australian Animal Welfare Act, National Institute of Health guidelines and ARRIVE guidelines. The present study and its procedures were approved by the Animal Ethics Committee of the Harry Perkins Institute for Medical Research, Perth, Australia (approval no. AE114) and the University of Western Australia, Perth, Australia (approval no. F71731). All handling, procedure and assistance techniques training was provided by the Bioresources team at the Harry Perkins Institute for Medical Research centre.
Not applicable.
GD is Wesfarmers Chair in Cardiology at the University of Western Australia with an Adjunct Professor appointment at UOHI. GD reports 3 paid lectures from AstraZeneca, Pfizer, and Amgen not related to the topic in the manuscript. GD provides consultancy services and also has an equity interest Artrya Pty Ltd. The other authors (BB, SL, HPL, TS, SV, VFCM and GW) have nothing to disclose and declare that they have no competing interests.
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.
FDG-PET images from TIGR4
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.
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.
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.
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)
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.