Background
Chronic obstructive pulmonary disease (COPD), including emphysema and chronic bronchitis, is the third leading cause of death in the US [
1]. COPD refers to a broad group of lung diseases with airflow limitation, parenchymal destruction, fibrosis around small airways involving several different cells (neutrophils, macrophage, CD8 lymphocytes) [
2,
3], and small-airway obstruction [
4]. The most studied of these conditions is emphysema, which is characterized by permanent inflammation and irreversible destruction of alveolar walls, leading to airspace enlargement and loss of elastic recoil and hyperinflation [
5]. Exposure to CS accounts for about 80 % of all COPD cases [
6‐
9], but other factors when combined with cigarette smoke play a role in exacerbating CS-induced COPD [
10,
11]. Repeated incidences of COPD exacerbations are associated with more rapid disease progression, poorer quality of life, and higher risk of mortality [
12,
13]. Environmental pollutants, including wood smoke [
10,
14,
15‐
17] and diesel exhaust [
18] play a role in enhancing CS-induced inflammation. Bacterial and viral infections are also major causes for COPD exacerbations [
19‐
25] leading to decline in lung function and concomitant acute deterioration in respiratory health [
12]. Interestingly, as many as 40 to 60 % of all exacerbations are attributed to respiratory viral infections alone [
26,
27].
Interactions between CS exposure and viral infection induce exaggerated inflammatory and tissue remodeling responses [
28,
29]. Viral infections, such as IAV [
30‐
32] and respiratory syncytial virus (RSV) [
19,
24,
25] are associated with acute COPD exacerbations, but the severity of infection in patients with COPD depends on both viral and host factors [
33]. For example, RSV accounts for a quarter of all viral-induced COPD exacerbations [
19,
24,
25] and is an important respiratory pathogen in the elderly, particularly in those with chronic lung diseases such as COPD [
23‐
25]. Approximately 60 million people currently smoke cigarettes in the US alone, and many of the immunocompromised elderly individuals are susceptible to RSV infections and will have recurrent seasonal flu infections. To understand disease progression and develop better therapies, establishing animal models that reflect these conditions in humans is crucial.
While the development of emphysema in mice requires an exposure to CS for 4–8 months [
34‐
38], infection with respiratory viruses enhances CS-induced inflammation and causes emphysema within 6 weeks [
28].
The purpose of the current study was to establish mouse models that recapitulate the cardinal features of COPD within a short period of time by comparing extent of inflammation and emphysema after a 4 week CS exposure in combination with a single infection of either IAV, RSV, or multiple instillation with Poly(I:C). We show that such a 2-hit system enhances lung inflammation and the development of emphysema. Despite these similarities, these animal models also showed major differences in the type and extent of inflammation and alveolar destruction.
Discussion
The present study utilized mouse models with existing CS exposure to compare extent of inflammatory responses and tissue destruction when infected with IAV, RSV, or instilled with Poly (I:C). When comparing these three models, one needs to consider the differences in the viral titers for IAV and RSV used to infect the mice. In addition, multiple doses of Poly (I:C) were delivered to mice over the last two weeks of CS exposure, and mice were euthanized right after 4 weeks of exposure to CS and were not kept for an additional 14 days in FA as in the cases with IAV and RSV groups. Repeated administration was chosen to compensate for the lack of replication in the poly(I:C) group.
We found that by day 4 post infection CS-exposed groups had significantly lower copy number of viruses than the FA groups for both IAV and RSV study groups. This may be due to early induction of type I IFN by cigarette smoke [
28,
55], which may suppress virus replication. As inflammation progresses, IFN response may wane resulting in delayed viral clearance and more titer in CS-injured lungs than FA-exposed lungs on day 14 post infection.
While the overall total numbers of inflammatory cells recovered in the BAL of CS-IAV/RSV and CS-poly(I:C) mice were synergistically increased, differences in the type and degree of inflammatory responses were observed among these groups. The CS-IAV group displayed increased number of macrophages and lymphocytes with few neutrophils, and CS-RSV group showed increased lymphocytes and neutrophils, similar to that observed in the CS-poly(I:C) group. These findings are consistent with previous studies showing that IAV or poly (I:C) lead to additive effects in the level of BAL inflammatory cells in mice exposed to CS [
28,
56]. Interestingly, despite higher titers used for RSV, IAV caused more inflammation suggesting that different viruses have distinct inflammatory patterns either alone or when combined with CS exposure. Macrophages and neutrophils are the main sources of proteases in lungs, and there are correlations between the degree of macrophage and neutrophil inflammation and severity of airflow obstruction [
57]. Although significant increases in the BAL neutrophils were observed among the models tested, poly (I:C) caused the mildest form of emphysema in CS-exposed mice. For the poly(I:C) group, emphysema measures were assessed at 4 weeks of CS exposure while the IAV and IAV groups of mice were assessed 14 d later. Further, IAV or RSV alone did not cause increases in alveolar spaces compared to their respective vehicle controls. Hence, it is possible that the poly(I:C) group could develop more pronounced structural changes in CS-exposed mice similar to the virally challenged groups given the appropriate time for the changes to develop. Emphysema that develops in mice after 6 months of exposure remains pronounced even 6 months later [
37], and future studies will need to examine the persistence of the observed emphysematous changes several months after the initial infections.
Both IAV and RSV infection induced IL-17a, IL-17b, IL-17d, and IL-17f, IL-1β, IL-12b, IL-18, IL-23, Ccl-2, and Ccl-7 mRNA levels in the lungs of CS-injured mice. CD8
+ T-lymphocytes are critical for the induction of inflammation and tissue destruction in a murine model of smoke-induced emphysema [
58]. CD4
+ T cells, upon activation and expansion, develop into different T helper cell subsets with different cytokine profiles and distinct effector functions [
59]. Thus, IAV or RSV in CS-exposed mice may cause the differentiation of Th17 cells or may affect γδ T cells, NK cells, and neutrophils to produce IL-17 in response to CS exposure dependent induction of IL-1β, IL-18, or IL-23 [
60]. Despite higher titer for RSV, IAV was more potent in increasing expression of IL-17a, IL-17c, IL-12b, IL-18, IL-23a, IL-1β, Ccl-2, and Ccl-7 than RSV. It is possible that cytokine expression was also increased in the poly(I:C) group, but emphysematous changes were not apparent. Although we did not compare cytokine in the lungs of this group of mice with the RSV and IAV groups, a study using CS-poly(I:C) mouse model showed that poly(I:C) was a potent stimulator of IL-18, IL-12/IL-23 p40, and type I and type II IFNs with significantly greater responses in mice exposed to CS compared with mice in FA. This study suggest that IL-18Rα and IFN-γ play critical role in the pathogenesis of the inflammation and remodeling that is induced by CS plus poly(I:C). IAV [
43,
61] and RSV [
62] have been shown to induce Th17-related cytokines. Future studies will elucidate the specific roles of the different cytokines and chemokines in IAV- or RSV-infected or poly(I:C) treated mice exposed to CS.
In mice, IL-17 is essential for the development of emphysema from long term CS exposure [
49] by activating the IL-17 → Ccl-2 → MMP-9 → MMP-12 signaling axis [
49]. Increases in IL-17-regulated genes and chemokines such as Ccl-2 and Ccl-7 are associated with COPD progression in humans [
63‐
65]. This is consistent with our data showing that increased IL-17 expression was associated with increased expression of MMP-12 mRNA levels. Mouse models with MMP-12-deficient macrophages [
51,
66] and pharmacological inhibition of MMP-12 [
66] showed that MMP-12 activity plays an important role in the development of emphysema.
It has long been believed that, in cigarette smoke–induced COPD, the alveolar destruction and enlargement is a direct consequence of inflammation and the associated imbalance in the extracellular matrix protease and antiprotease response, which leads to degradation of the elastin [
67]. However, despite differences in inflammatory responses, IAV and RSV infections led to similar extent of emphysema in the CS-exposed mice. Further, IAV and RSV infections significantly enhanced TUNEL positivity and upregulation of the MMP-12 mRNA to a similar extent. Also, despite increases in inflammation by poly (I:C) similar to RSV, extent of emphysema caused by poly (I:C) was milder. These findings suggests that inflammation does not necessarily correlate with lung pathology as was shown for CS-induced emphysema in C3H and C57Bl/6 mice [
11]. It is also possible that regardless of extent of lung inflammation, the severity of alveolar destruction and airspace enlargement in mice remains mild [
37], likely because of lung structure or length of chronic inflammation. While infection with RSV leads to differentiation of Th17 cells [
62,
68] and is associated with skewing the immune system away from the Th1 response [
69], poly (I:C) may cause more Th1 than Th17 differentiation [
70,
71]. Furthermore, although poly (I:C) activates TLR3 [
72], repeated doses of poly (I:C) induce inflammation and alveolar remodeling via pathways independent of TLR3 [
28]. Future studies will investigate the role of innate versus Th17 immunity in the pathogenesis of IAV-, RSV-, and poly (I:C)-induced lung inflammation in CS-exposed mice.
Both weighted mean alveolar volume and Lm are useful parameters of peripheral lung structure and have utility in studying experimental emphysema [
73,
74]. However, Lm reflects the dimensions of alveoli but is not a direct estimate of alveolar diameter or mean alveolar size. Although weighted mean alveolar volume,
Lm, and indexes of lung elasticity are correlated in excised non-diseased lungs of humans [
75] and several animal species [
76] Lm may not be the most accurate measure. Lm is a function of lung volume [
76,
77] but may not be able to separate the effects of tissue destruction from those on tissue distension and from any changes in lung elasticity that results in changes in lung volume, as would be expected with emphysema. Thus, conclusions drawn from Lm alone should be interpreted with caution.
Abbreviations
BAL, broncho-alveolar lavage; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; ES&H, Environmental Safety and Health department; FA, filtered air; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; IACUC, Institutional Animal Care and Use Committee; IAV, Influenza A Virus; Lm, mean linear intercept; mRNA, messenger ribonucleic acid; PCR, polymerase chain reaction; Poly(I:C), polyinosine-polycytidylic acid; RSV, respiratory syncytial virus; RT-PCR, reverse transcriptase polymerase chain reaction; SEM, standard error of the mean; TPM, total particulate matter; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.