Influenza A virus-dependent remodeling of pulmonary clock function in a mouse model of COPD

Abstract

Daily oscillations of pulmonary function depend on the rhythmic activity of the circadian timing system. Environmental tobacco/cigarette smoke (CS) disrupts circadian clock leading to enhanced inflammatory responses. Infection with influenza A virus (IAV) increases hospitalization rates and death in susceptible individuals, including patients with Chronic Obstructive Pulmonary Disease (COPD). We hypothesized that molecular clock disruption is enhanced by IAV infection, altering cellular and lung function, leading to severity in airway disease phenotypes. C57BL/6J mice exposed to chronic CS, BMAL1 knockout (KO) mice and wild-type littermates were infected with IAV. Following infection, we measured diurnal rhythms of clock gene expression in the lung, locomotor activity, pulmonary function, inflammatory, pro-fibrotic and emphysematous responses. Chronic CS exposure combined with IAV infection altered the timing of clock gene expression and reduced locomotor activity in parallel with increased lung inflammation, disrupted rhythms of pulmonary function, and emphysema. BMAL1 KO mice infected with IAV showed pronounced detriments in behavior and survival, and increased lung inflammatory and pro-fibrotic responses. This suggests that remodeling of lung clock function following IAV infection alters clock-dependent gene expression and normal rhythms of lung function, enhanced emphysematous and injurious responses. This may have implications for the pathobiology of respiratory virus-induced airway disease severity and exacerbations.

Figure 1. Influenza A virus infection differentially affects body weight, mortality and behavior in chronic air or CS-exposed mice.

Data from uninfected air- and CS-exposed mice from a previous experiment were included for comparison [panels c–e;30]. (a) Chronic CS-exposed mice infected with IAV showed a modest reduction in weight, marked by a decline on days 1–3 post-infection relative to air-exposed mice infected with IAV. (b) Mortality was monitored for 9 days post IAV infection. Data are representative of mean ± SEM (n = 30–40 mice/group). ** P < 0.01 significant compared to Air+Virus group. Analysis of differences in survival over time was determined by a Mantel-Cox test (P < 0.68). (c) Nocturnal activity for 9 days post-infection was plotted as ambulatory counts. IAV infection reduced locomotor activity in CS-exposed mice by 70–80% during days 1–7 post-infection when compared to CS-exposed mice treated with saline. Similarly, IAV infection reduced locomotor activity in CS-exposed mice by 70–80% on days 1–2 and day 4 post-infection but only 30–45% during days 5–9 post-infection when compared to Air+Virus treated mice. Data are mean ± SEM (n = 6 mice/group) for each time point. ** P < 0.01; *** P < 0.001 significant compared to control groups (air or Air+Virus); ^$$ P < 0.01 significant compared to air-exposed mice; ^# P < 0.05; ^{# #} P < 0.01; ^{# # #} P < 0.001 significant compared to CS-exposed mice. (d) Representative double plotted actograms of total cage activity from chronic air, chronic CS, Air+Virus and CS+Virus treated mice. In panel d, gray shading indicates the dark phase (ZT12-24) and activity was not recorded during body weight measurements (ZT5-6). Red arrow head indicates day 0 IAV infection and asterisks denotes the time during which the mice were infected with IAV (ZT4-6). (e) Periodogram analysis of activity in L:D during days 1–8 post IAV infection. Period was very similar between air and CS groups, whereas the Air+Virus and CS+Virus groups showed increased variation in period though the mean was not significantly different from controls. Data are mean ± SEM (n = 6 mice/group).

Figure 2. Influenza A virus infection differentially affects body weight, mortality and behavior in wild-type (WT) and BMAL1 KO mice.

(a) IAV infection significantly reduced body weight of both WT and BMAL1 KO mice on day 2–9 post-infection compared to uninfected controls. * P < 0.05; ** P < 0.01; *** P < 0.001 significant compared to WT-Saline or BMAL1 KO-Saline; (b) Mortality was increased following IAV infection in both WT and BMAL1 KO mice, reaching 100% in BMAL1 KO mice within 9 days post-infection. Data are representative of mean ± SEM (n = 10 WT-Saline; n = 4 BMAL1 KO-Saline; n = 13 WT-Virus; n = 13 BMAL1 KO-Virus). Survival over time was analyzed with a Mantel-Cox test (P < 0.001). (c) Nocturnal activity of IAV infected mice (both WT and BMAL1 KO) was reduced on days 1–8 post-infection when compared to uninfected controls. ***P < 0.001 significant compared to uninfected controls; ^$ P < 0.05; ^$$ P < 0.01 significant compared to WT-Saline treated mice; ^†† P < 0.01 significant compared to WT-Virus infected mice. (d) Representative double plotted actograms showing considerable reduction in total cage activity of IAV infected WT and BMAL1 KO mice relative to uninfected controls. In panel d, gray shading indicates the dark phase (ZT12-24) and activity was not recorded during body weight measurements (ZT5-6). Red arrow head indicates day 0 IAV infection and asterisks denotes the time during which the mice were infected with IAV (ZT4-6). (e) Periodogram analysis of activity in L:D was calculated during days 1–8 post-infection. Though there was no overall effect of IAV infection on period in either group an increase in variation was detected following infection in BMAL1 KO mice. Data are mean ± SEM (n = 10 WT-Saline; n = 13WT-Virus; n = 4 BMAL1 KO-Saline; n = 13 BMAL1 KO-Virus).

Figure 3. Diurnal rhythms of clock gene expression in the lungs are differentially affected by chronic CS and IAV infection.

Data from uninfected air-exposed mice from a previous experiment were included for comparison [panels a-b;30]. Lung tissues were harvested every 6 h for 24 h beginning at ZT0 day 9 post-infection. (a) Expression of core clock genes (bmal1, clock, per1, cry1, and rev-erbα) in mouse lung tissue. CircWave analysis confirmed statistically significant rhythms of clock gene expression in Air+Virus (P < 0.05 for per1 and cry1; P < 0.001 for bmal1) and CS+Virus (P < 0.05 for per1; P < 0.01 for bmal1) treated mice. (b) IAV infection adjusted the phase of clock gene expression in a gene- and treatment- (Air vs. CS) dependent manner. Center of gravity (COG) or peak phase for each clock gene was plotted on a horizontal phase map. In panels a and b, gray shading indicates the relative dark phase (ZT12-24). Data from air-exposed (open circle), Air+Virus (gray square) and CS+Virus (solid diamond) mice are representative of mean ± SEM (n = 3-4 mice/group) for each time point. * P < 0.05; ** P < 0.01; *** P < 0.001 significant compared to Air group. ^{# #} P < 0.01; significant compared to Air+Virus group. (c) Effect of CSE and IAV infection on the amplitude and period of PER2::LUC expression in lung tissue explants. PER2::LUC expression in representative lung tissue explants treated with medium alone (control), 0.1% CSE, virus alone (IAV, 300 HAU/ml), and 0.1% CSE+Virus. Treatment at the time of culture with 0.1% CSE and 0.1% CSE+Virus dampened the rhythm of PER2::LUC expression in lung tissue. Treatment with 0.1% CSE had a small effect whereas IAV infection significantly increased the period of PER2::LUC expression in lung explants. This effect was attenuated in explants treated with 0.1% CSE and IAV. Different colored traces represent tissue explants from different animals. Data are representative of mean ± SEM (n = 12-15/group). * P < 0.05 significant compared to control group; ^# P < 0.05 significant compared to Virus group.

Figure 4. Chronic CS-exposed mice infected with influenza A virus show increased inflammatory cell influx and proinflammatory cytokine release in BAL fluid.

Data from chronic (6 months) air- or CS-exposed mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Data from uninfected air- and CS-exposed mice from a previous experiment were included for comparison [panels a–d;30]. The total number of inflammatory cells was determined in BAL fluid from air, CS, Air+Virus and CS+Virus infected mice on day 9 post-infection. At least 500 cells in the BAL fluid were counted to determine (a) total cells, (b) total macrophages, (c) total lymphocytes and (d) total neutrophils. Data are representative of mean ± SEM (n = 3-4 mice/group) for each time point. * P < 0.05, *** P < 0.001 significant compared to air-exposed mice; ^$ P < 0.05 significant compared to air-exposed mice; ^{# # #} P < 0.001 significant compared to CS-exposed mice. Levels of proinflammatory mediators (e) MCP-1, (f) MIP-2 and (g) IL-6 levels were measured in BAL fluid obtained from air, CS, Air+Virus and CS+Virus infected mice. IAV infection of chronic CS-exposed mice alters diurnal rhythms of proinflammatory cytokine release in mouse lungs. Data are representative of mean ± SEM (n = 3-4 mice/group) for each time point. * P < 0.05; ** P < 0.01; *** P < 0.001 significant compared to air or Air+Virus groups; ^# P < 0.05; ^{# #} P < 0.01; ^{# # #} P < 0.001 significant compared to CS-exposed mice.

Figure 5. Chronic CS-exposed mice infected with IAV show persistent inflammation, mucus hypersecretion, and pulmonary fibrosis.

Data from chronic (6 months) air- or CS-exposed mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Lungs were harvested on day 9 post-infection. (a) Representative images of lung tissues stained with hematoxylin and eosin (H&E) to demonstrate parenchymal and bronchial airway inflammation. Bronchial inflammation scores were calculated for each treatment group. (b) Representative images of lung tissues stained with Periodic-acid Schiff (PAS) to visualize mucus overproduction induced by IAV in the bronchial epithelium of chronic air- and CS-exposed mice. Average mucus scores from 3–4 different areas per slide/treatment group (n = 4-5mice/group) was used to calculate the percentage of PAS positive cells. (c) Representative images of lung tissues stained with Gomori’s Trichrome to visualize matrix accumulation/collagen deposition and quantified by Ashcroft fibrosis score. Original magnification x200. Data are representative of mean ± SEM (n = 4-5 mice/group). * P < 0.05; ** P < 0.01; significant compared to air- or CS-exposed mice. ^{# #} P < 0.01; ^{# # #} P < 0.001; significant compared to air-exposed mice.

Figure 6. Daily rhythms of lung function are differentially affected by chronic CS-exposure and influenza A virus infection.

Data from chronic (6 months) air- or CS-exposed WT mice given intranasal inoculation of either saline (control group) or influenza A virus (IAV; treatment group) at ZT4-6 are shown. Data from uninfected air-exposed mice from a previous experiment were included for comparison [panels a–b;30]. (a) Daily rhythms of compliance, resistance and elastance were measured in air, Air+Virus and CS+Virus mice. As in previous experiments, measurements were taken on day 9 post-infection. (b) COG or peak phase values for each measure of lung function were plotted on a horizontal phase map. Gray shading in panels a and b indicates the relative dark phase (ZT12-24). Data from air-exposed (open circle), Air+Virus (gray square) and CS+Virus (solid diamond) are representative of mean ± SEM (n = 3-4 mice/group) for each time point. ** P < 0.01; *** P < 0.001, significant compared to Air+Virus; ^$ P < 0.05; ^$$$ P < 0.001 significant compared to air-exposed mice. (c) Influenza A virus infection altered lung function in WT mice. After day 9 post-infection, lung compliance, resistance and tissue elastance were determined in WT-Saline, WT-Virus, BMAL1 KO-Saline and BMAL1 Het-Saline (heterozygous) treated mice. Data are representative of mean ± SEM (n = 10 WT-Saline; n = 8 WT-Virus, n = 4 BMAL1 KO-Saline and n = 6 BMAL1 Het-Saline) for each time point. *** P < 0.001, significant compared to WT-Saline; ^# P < 0.05; ^{# #} P < 0.01 significant compared to WT-Virus.