Background
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death world-wide, and the prevalence of COPD is increasing globally [
1]. COPD is characterized by airflow limitation that is progressive and is usually irreversible. Small airway remodeling, including narrowing of airways due to peribronchiolar fibrosis, goblet cell metaplasia and excessive mucus production, is now accepted as an important cause of airflow obstruction in COPD [
2]. In addition, lung parenchyma is destroyed by proteolytic damage (emphysema), reducing the elasticity and gas-exchange surface area of the lung. Acute exacerbations further enhance airways obstruction and accelerate progression of lung disease in these patients (reviewed in [
3]). However, the underlying mechanisms for these changes are not well understood, partly because small-animal models do not recapitulate all the typical features of human COPD.
COPD has been modeled in mice by administration of proteases, lipopolysaccharide (LPS), chemicals and cigarette smoke (CS). In particular, CS has been used extensively to investigate mechanisms of COPD pathogenesis since it is the major risk factor in the development of COPD [
3]. While whole body exposure to CS for a short-term (3 days to 4 weeks) has been useful in evaluating the mechanisms of CS-induced acute lung inflammation and defective innate immune responses to subsequent infections [
4‐
7], long-term exposure to CS for periods up to 6 months has been employed to understand the mechanisms of emphysema development [
8‐
11]. However, neither of these models show changes in small airways, which plays a major role in the development of airflow limitation in COPD implying that other factors in addition to CS are required to mimic COPD lung disease in mice [
12].
Although CS exposure is the key insult in the pathogenesis of COPD, only 25 to 35% of smokers develop COPD [
13,
14] suggesting the contribution of other factors, such as genetic background, concurrent respiratory infections, and aberrant host responses in the development of COPD. Respiratory pathogens including bacteria, viruses and fungi are often present in the airways of COPD patients and therefore it is plausible that respiratory pathogens or their products such as enterotoxin, endotoxin, viral RNA may contribute to disease pathogenesis [
15‐
17]. For example Kang
et al. demonstrated that CS synergizes with synthetic double stranded (ds) RNA, a viral RNA mimetic to induce enhanced inflammatory and emphysematous changes, and airway fibrosis in the mouse lungs [
17]. However these mice did not develop goblet cell metaplasia, increased mucus production or airways obstruction, which are also important features of COPD. Similar results were observed when mice were infected with influenza virus instead of treating with dsRNA. Enterotoxin B isolated from
S. aureus was shown to exacerbate CS-induced inflammatory changes in mouse lungs and induce goblet cell metaplasia and formation of lymphoid aggregates, but these mice did not develop emphysema. Chronic exposure of mice to lysates of non-typeable
H. influenzae (NTHi) was shown to induce airway inflammation but not emphysema or airway remodeling [
18]. Endotoxin, a bacterial cell wall component is present in abundant amounts as a contaminant in CS [
19,
20] and prolonged intratracheal exposure of mice to endotoxin, (twice week for 3 months) induces lung inflammation, and changes in both parenchyma and airways which persists up to 8 weeks [
21]. We demonstrated that exposure of mice once a week to combination of elastase and endotoxin for 4 weeks induces all the features of COPD [
22]. The latter three models although exhibit features of COPD and indicate involvement of bacterial factors in COPD pathogenesis, these models may not be representative of CS-induced changes. Based on these observations, in the present study we examine a novel concept that in addition to CS, exposure to bacteria is required to induce typical features of COPD including emphysema, airway remodeling, and lung inflammation in mice. Since NTHi is frequently isolated from clinically stable COPD patients as well as during exacerbations [
23], we used NTHi in the present study. We also evaluated susceptibility to rhinovirus (RV) infection in this model, because RV is associated with virally mediated exacerbations of COPD and sometimes leads to progression of lung disease [
24,
25].
Discussion
Exacerbations in COPD patients are characterized by increased inflammation and increased lower respiratory tract symptoms requiring change in therapy such as treatment with antibiotics, steroids or antiviral drugs [
33]. Exacerbations may be triggered primarily by viral or bacterial infections and are often associated with accelerated progression of lung disease. RV, which causes self-limiting infections in healthy individuals is responsible for the majority of virus-related exacerbations in patients with COPD [
24,
25], but the underlying mechanisms are not well understood. In this study, we developed a mouse model which displays phenotypic characteristics of COPD, including emphysema, mild but diffuse lung inflammation, and goblet cell metaplasia. We combined this model with our previously described mouse model of RV infection [
22,
34] and show that these mice show prolonged neutrophilic lung inflammation and airways obstruction, similar to that observed in mild COPD patients experimentally infected with RV [
35], indicating the suitability of this model to elucidate mechanisms of COPD exacerbations.
The mouse model of COPD developed in this study supports our primary hypothesis that both CS and bacteria are required for development of COPD–like changes in mice and also render mice susceptible to viral infection. We demonstrate that although CS is the major risk factor in the development of COPD, mice exposed to CS alone for 8 weeks only develop mild lung inflammation and emphysema but not small airway disease which is consistent with previous observations [
4,
8‐
11]. In contrast, exposure of mice to a combination of CS and NTHi induces not only more pronounced emphysema and lung inflammation than mice exposed to CS alone, but also goblet cell metaplasia in the airways, which is one of the pathologic features of COPD. In addition, mice exposed to combination of CS/HK-NTHi also show heightened susceptibility to viral infection with further increases in lung inflammation and goblet cell metaplasia. Together, these findings suggest that this novel mouse model of COPD is not only useful in understanding the mechanisms of exacerbations, but also for delineating the mechanisms underlying progression of lung disease in COPD. However, it should be noted that continuous exposure to CS and/or intermittent infection with bacteria or virus may be required to maintain chronicity of the COPD-like changes in these mice.
NTHi is one of the more commonly isolated organisms from clinically stable COPD patients and also at exacerbations [
23], and patients who are chronically colonized with NTHi show significantly more neutrophilic airway inflammation than those who are not colonized [
15,
36]. Further, repeated exposure to extracts of heat-killed NTHi causes airway inflammation in mice with cellular and cytokine profiles similar to COPD [
18]. Finally, NTHi products increase mucin gene expression
in vivo and
in vitro [
37]. These observations indicate that NTHi may contribute to the development of COPD particularly, small airway disease and lung inflammation. Based on these facts, we postulated that NTHi may synergize with CS to induce development of COPD-like features in mice which includes changes in both small airways and parenchyma. In the present study, we opted to use low-dose (5 × 10
6 CFU) heat-killed NTHi to induce milder and sustained inflammation with minimal infiltration of neutrophils as opposed to acute neutrophil-dominated inflammation induced by live NTHi [
27,
28], to prevent extensive lung damage. The bacterial dose was chosen based on our preliminary experiments, in which mice were treated with heat-killed bacteria equivalent to 5 × 10
5, 5 × 10
6 or 5 × 10
7 CFU once a week for 4 consecutive weeks and sacrificed 4 weeks after the last treatment. By morphology, mice treated with 5 × 10
5 CFU did not show any detectable changes in the lungs and were very similar to untreated mice. In contrast, mice treated with 5 × 10
7 CFU showed severe lung inflammation, consolidation of parenchyma and pronounced goblet cell metaplasia in both small and large airways. Mice treated with 5 × 10
6 CFU showed mild to moderate lung inflammation with mild goblet cell metaplasia. In addition, we also found that two exposures to 5 × 10
6 CFU instead of four exposures were sufficient to induce these changes in the lung. Combination of this HK-NTHi treatment with CS however, led to more pronounced lung inflammation and goblet cell metaplasia, increased mucin gene expression and also the development of emphysema. Interestingly these mice also showed thickening of airway epithelia, similar to the airway epithelial hyperplasia observed in COPD patients [
38]. We also observed aggregates of inflammatory cells particularly in the peribronchiolar and perivascular areas resembling lymphoid aggregates [
16], but the nature of these aggregates is yet to be determined. We speculate that these pronounced pathological changes in CS/HK-NTHi-exposed mice may be the result of exaggerated host innate immune responses to bacterial products, in the presence of CS.
Alveolar macrophages in the lungs play an important role in clearing bacteria and limit bacteria-induced inflammation. CS has been demonstrated to affect the function of alveolar macrophages by shifting their phenotype from M2 to M1 [
39,
40]. M1 macrophages respond to bacterial antigens such as LPS by producing relatively more inflammatory cytokines than M2 macrophages [
41]. Although this is required for clearance of bacteria, sustained production of inflammatory cytokines may increase lung inflammation, emphysema and also induce goblet cell metaplasia [
8,
42]. Based on these observations, we speculate that CS-induced shift in macrophage phenotype may be one of the mechanisms by which CS synergizes with NTHi to increase expression of inflammatory cytokines and this in turn may lead to increased lung inflammation, emphysema and goblet cell metaplasia. Bacterial pathogens or their products other than NTHi may also induce COPD-like changes when combined with CS, but the magnitude of pathological changes may vary depending on the bacteria used. However, further studies are required to confirm this notion.
CS/HK-NTHi-exposed mice displaying the COPD phenotype were also found to be susceptible to subsequent infection with RV, a virus associated with a majority of viral-associated COPD exacerbations [
35,
43]. Four days after challenging with RV, CS/HK-NTHi-exposed mice despite showing minimally higher viral loads than similarly infected RA-, CS-, or NTHi-exposed mice, displayed sustained neutrophilia and lymphocyte infiltration, and increased expression of KC, MIP-2 and IP-10, which was not observed in mice from other groups. These changes were associated with progression in goblet cell metaplasia in small airways and increased expression of
Muc5AC (mucin gene) and
Gob5, a chloride channel that plays a role in mucin secretion [
32]. These mice also showed sustained airways hyperresponsiveness to methacholine challenge. Previously, we have shown that airway epithelial cells isolated from COPD patients are more susceptible to RV infection despite expressing interferon and other antiviral genes [
44]. This is probably due to increase in the number of goblet cells in COPD cell cultures, because previously goblet cells have been shown to be more permissive for RV infection [
45]. Therefore it is plausible that CS/HK-NTHi mice which show increased numbers of goblet cells in their airways may be more susceptible to RV infection. In addition, exposure to CS may also support replication of virus and induce aberrant production of inflammatory cytokines. Consistent with this notion, acute exposure to CS was shown to increase production of pro-inflammatory cytokines and reduce expression of antiviral genes in response to RV infection in airway epithelial cells [
46,
47]. Such aberrant increases in inflammatory cytokines following RV infection may further enhance lung inflammation, goblet cell metaplasia and airways obstruction. However further studies are required to confirm this notion. These observations are consistent with human studies, in which mild COPD patients were shown to develop sustained lower respiratory tract symptoms and increased infiltration of neutrophil and lymphocyte in the lungs after experimental rhinovirus infection [
35]. These changes correlated with decreases in lung function in these patients. Another noteworthy observation is that CS/HK-NTHi-exposed mice infected with RV also showed sustained increases in IP-10 levels, which has been proposed to be one of the biomarkers of COPD exacerbations [
48,
49]. These similarities in responses to RV infection between COPD patients and CS/HK-NTHi mice imply that this model may be useful in understanding the molecular mechanisms related to viral-associated COPD exacerbations and progression of lung disease.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SG, designed and conducted the experiments and analyzed the data; ATC and BK, provided technical support; PM and JMB, contributed intellectually; US, conceived and designed the study and wrote the manuscript. All authors read and approved the final manuscript.