Interventional low-dose azithromycin attenuates cigarette smoke-induced emphysema and lung inflammation in mice

Abstract

Cigarette smoke (CS)-induced emphysema is an important contributor to chronic obstructive pulmonary disease (COPD). We have shown the efficacy of azithromycin in reducing airway inflammation in COPD and in reducing exacerbations in severe asthma; however, the effects of long-term azithromycin on emphysema development have not been shown. We employed live animal imaging to monitor emphysema-like development and the effects of interventional azithromycin treatment in CS-exposed mice. BALB/c mice (female, 10 weeks; n = 10) were exposed to CS for 1 hr twice daily, 5 days/week, and for 12 weeks (CS). Half were cotreated with low-dose azithromycin during weeks 7-12 (CS + Azi; 0.2 mg kg^-1 day^-1 ). Microcomputed tomography (CT) and magnetic resonance imaging (MRI) scans were acquired longitudinally. Histological examinations were performed post mortem (mean linear intercept (Lm) and leukocyte infiltration). CS increased median Lm (CS: 42.45 µm versus control: 34.7 µm; p = .0317), this was recovered in CS + Azi mice (33.03 µm). Average CT values were reduced in CS mice (CS: -399.5 Hounsfield units (HU) versus control: -384.9 HU; p = .0286) but not in CS + Azi mice (-377.3 HU). CT values negatively correlated with Lm (r = -.7972; p = .0029) and T₂ -weighted MRI (r = -.6434; p = .0278). MRI also showed significant CS-induced inflammatory changes that were attenuated by azithromycin in the lungs, and positively correlated with Lm (r = .7622; p = .0055) and inflammatory foci counts (r = .6503; p = .0257). Monitoring of emphysema development is possible via micro-CT and MRI. Interventional azithromycin treatment in CS-exposed mice attenuated the development of pulmonary emphysema-like changes.

Keywords: COPD; chronic obstructive pulmonary disease; emphysema; in vivo imaging; magnetic resonance imaging; micro-CT; mouse model.

FIGURE 1

Emphysematous changes in CS‐exposed mice via micro‐CT analysis of the lower lung are attenuated by azithromycin. (a) Percentage changes in mouse weight over time. (b) Representative micro‐CT image slice of a mouse lung prior to processing (left) and postthresholding (right). (c) Time course of average Hounsfield unit (HU) values of the lower lungs (mean ± SD). (d) Average HU values at week 6. (e) Average HU values at week 12 prior to postmortem. (f) Correlation of week 12 average HU values with Lm. Data presented as median ± range (unless otherwise stated). * represents significance between cohorts, p < .05. Dotted lines show the 95% confidence interval

FIGURE 2

Assessment of emphysema‐like changes in the lungs. (a–c) Representative H&E‐stained lung sections at 12 weeks. (a) control, (b) CS‐exposed (CS), and (c) CS‐exposed + azithromycin treatment (CS + Azi). Scale bar is 250 µm. (d) Mean linear intercept (Lm) values were taken as a measure of alveolar size and emphysematous changes. Data represent the average Lm based on the measurements taken from 3 lung sections per mouse; median ± range. n = 4–5 mice/group. * represents significance between cohorts, p < .05; ** indicates p < .01

FIGURE 3

Inflammation in the lungs and kidneys of CS‐exposed mice is attenuated by azithromycin. (a) Representative coronal, T₂‐weighted MR images of live mice. (b and c) Comparison of T₂ intensity in the lungs and kidneys. (d) Correlation of T₂‐weighted MRI intensity values of the lung and average CT value (HU). (e) Correlation of T₂‐weighted MRI intensity values of the lung and Lm. Data presented as median ± range. Dotted lines show the 95% confidence interval. * represents significance between cohorts, p < .05. Dotted lines show the 95% confidence interval. n = 4 mice/group

FIGURE 4

CS‐exposed mice showed increased leukocyte infiltration into lung tissue that was attenuated by azithromycin. (a and b) H&E‐stained lung sections from CS‐exposed mice showing examples of leukocyte foci (white arrows) (a) near blood vessels (BV) and (b) airway epithelia (Epi). (c) Fold change in the number of leukocyte foci in the lungs. (d) Correlation between T₂‐weight MRI intensity values and leukocyte foci in the lung. Data presented as median ± range. Dotted lines show the 95% confidence interval. * represents significance between cohorts, p < .05; ** indicates p < .01. Dotted lines show the 95% confidence interval. n = 4–5 mice/group