Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis

Abstract

Background: Viral bronchiolitis is the leading cause of hospitalization in young infants. It is associated with the development of childhood asthma and contributes to morbidity and mortality in the elderly. Currently no therapies effectively attenuate inflammation during the acute viral infection, or prevent the risk of post-viral asthma. We hypothesized that early treatment of a paramyxoviral bronchiolitis with azithromycin would attenuate acute and chronic airway inflammation.

Methods: Mice were inoculated with parainfluenza type 1, Sendai Virus (SeV), and treated daily with PBS or azithromycin for 7 days post-inoculation. On day 8 and 21 we assessed airway inflammation in lung tissue, and quantified immune cells and inflammatory mediators in bronchoalveolar lavage (BAL).

Results: Compared to treatment with PBS, azithromycin significantly attenuated post-viral weight loss. During the peak of acute inflammation (day 8), azithromycin decreased total leukocyte accumulation in the lung tissue and BAL, with the largest fold-reduction in BAL neutrophils. This decreased inflammation was independent of changes in viral load. Azithromycin significantly attenuated the concentration of BAL inflammatory mediators and enhanced resolution of chronic airway inflammation evident by decreased BAL inflammatory mediators on day 21.

Conclusions: In this mouse model of paramyxoviral bronchiolitis, azithromycin attenuated acute and chronic airway inflammation. These findings demonstrate anti-inflammatory effects of azithromycin that are not related to anti-viral activity. Our findings support the rationale for future prospective randomized clinical trials that will evaluate the effects of macrolides on acute viral bronchiolitis and their long-term consequences.

Figure 1

Azithromycin attenuated viral-dependent weight loss. (A) Experiment time line. Seven-week-old C57BL/6J female mice were inoculated with Sendai virus 5,000 egg infectious dose 50% (SeV 5 K). Mice were treated daily with PBS or azithromycin day 0 (one hour after SeV inoculation) through day 7. On days 8 and 21, bronchoalveolar lavage (BAL) fluid and lungs and were harvested. (B) Percentage of weight change from baseline (day 0) in PBS (black square) versus azithromycin (black triangle) treated mice. Values are the mean ± SEM (n = 23 in each group). A significant decrease between PBS and azithromycin treatment is indicated (*, p < 0.05). (C) Kaplan-Meier analysis of survival. No statistical difference between treatment groups (n = 23 in each group) was determined by log-rank test.

Figure 2

Azithromycin attenuated viral-dependent airway inflammation. Mice were inoculated with SeV and treated as in Figure 1. Eight days post-viral inoculation, lung sections were obtained from naive mice (Naive, left column); SeV infected mice treated with PBS (SeV + PBS, middle column), and SeV infected mice treated with azithromycin (SeV + Azithro, right column). Representative photomicrographs of hematoxylin and eosin stained lung sections are shown (n = 11, Azithro; n = 12, PBS). Inflammatory cells within the airway are indicated (arrow). Bar = 50 μm (top) and 25 μm (bottom).

Figure 3

Azithromycin attenuated viral-dependent accumulation of total cells, macrophages, lymphocytes and neutrophils in the BAL. BAL from mice treated as in Figure 1 was analyzed for total and differential cell number eight days post-inoculation. Groups are labeled as in Figure 2A and values are the mean ± SEM (n = 11, Azithro; n = 12, PBS) of total BAL cells (A), macrophages (B), lymphocytes (C) and neutrophils (D). A significant decrease between PBS and azithromycin treatment is indicated (*, p < 0.05).

Figure 4

Azithromycin attenuated viral-dependent airway inflammation is associated with decreased concentrations of BAL inflammatory mediators. BAL from mice treated as in Figure 1 was analyzed for inflammatory mediators eight days post-inoculation. Concentrations of inflammatory mediators in the cell-free BAL supernatant were determined using a multiplex flow-cytometry based assay (Bio-Rad Laboratories). Groups are labeled as in Figure 2B and values are the mean ± SEM (n = 11, Azithro; n = 12, PBS) of G-CSF (A), CCL2/JE (B), CCL4/MIP-1β (C), CCL5/RANTES (D). A significant decrease between PBS and azithromycin treatment is indicated (*, p < 0.05).

Figure 5

Azithromycin attenuated viral-dependent airway inflammation is independent of Sendai virus load. Mice were inoculated with SeV and treated as in Figure 1. Five and eight days post-inoculation, whole-lung RNA was analyzed for Sendai virus-specific and GAPDH RNA by one-step fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR). The mean of duplicate measurements of SeV-specific RNA was normalized to GAPDH and reported as the SeV to GAPDH ratio. Values are the mean ± SEM (n = 6, day 5; n = 4, day 8).

Figure 6

Azithromycin treatment attenuated chronic viral-dependent airway inflammation. Mice were inoculated with SeV and treated as in Figure 1. Twenty-one days post-inoculation, lung sections and BAL fluid were harvested. Values are the mean ± SEM (n = 10, Azithro; n = 8, PBS) of total BAL cells (A), macrophages, lymphocytes and neutrophils in the BAL (B), and concentrations of the chemokines: G-CSF (C) and CCL1/KC (D). A significant decrease between PBS and azithromycin treated mice is indicated (*, p < 0.05).