Chapter 57A: Smoke Exposure Models in COPD

Chronic obstructive pulmonary disease (COPD) is defined as a disease state characterized by airflow limitation that is not fully reversible.¹ The limitation in airflow is caused by airway inflammation,² loss of lung elasticity,³ lung tissue destruction,⁴ and the closure of small airways.^5,6 The airflow obstruction progresses over time and loss of lung function impairs the ability of individuals to carry out routine daily activities and greatly increases their risk of death. Indeed, COPD is now the third leading cause of death in the United States and is projected to become the third leading cause worldwide within the next 20 years.^7,8 While the age-adjusted mortality for cardiovascular diseases had decreased significantly over the past 3 decades, the age-adjusted mortality for COPD has increased over this time period⁹ highlighting the need for better therapies^10,11 and increased COPD research. It is well known that exposure to cigarette smoke, both first and second hand, is the primary etiologic factor associated with this disease. Although great strides have been made in reducing smoking prevalence in the United States, 43.8 millions people or 19.0% of the United States population (age 18 or older) continue to smoke.¹² Moreover, smoking remains a major public health issue for adolescents with the latest surveys showing that 16% of all eighth graders had tried smoking and 17% of high school students continue smoking beyond graduation.¹³ Internationally, the picture is even bleaker with a smoking prevalence of 28% in China,¹⁴ 27% in Germany, and 36% in Russia.^15,16 These figures ensure that this disease will be a major public health issue for the foreseeable future.

While cigarette consumption is the main risk factor for COPD, 10% to 15% of COPD cases are not related to cigarette smoke exposure.¹⁷ In the developing world, it is estimated that air pollution from biomass smoke accounts for 2.2 to 2.5 million deaths annually.¹⁸ Epidemiologic studies have implicated biomass use in the development of chronic obstructive pulmonary disease (COPD) in adults and acute lower respiratory infection in children.^19,20 Women are particularly affected given their daily usage of these fuel sources for cooking. Moreover, exposure in women begins early in life and continues for decades.²¹ Indeed, several studies have found increased markers of inflammation and oxidative stress in premenopausal women exposed to biomass smoke.^22,23,24 Worldwide, it is estimated that 3 billion people utilize biomass as their primary source of domestic energy.²⁵ Though plans are underway to distribute improved cook stoves that emit less harmful particulates, these efforts will take time and a continued commitment on the part of public health authorities to impact on the development of COPD in these impoverished communities. Data obtained from cigarette smoking studies suggest that despite discontinuing smoking the rate of progression of COPD does reduce, but it does not prevent persistent airway inflammation and significant progression of COPD observed by CT scan.² Therefore, a large percentage of the current 3 billion people utilizing biomass sources may already have irreversible inflammation and COPD.

Acute episodes of respiratory distress in patients with COPD account for up to 5% to 10% of all emergency medical admissions²⁶ and almost 10% of these patients are referred to the ICU for further management.²⁷ Hospital mortality for those admitted with COPD is 5.6% and approximately 18% of COPD subjects will die within 180 days posthospital discharge. Historically, COPD patients that develop respiratory failure without a precipitating cause (pneumonia, pneumothorax, pulmonary embolism) have been perceived to have very poor outcomes. Indeed, in the United Kingdom, patients with COPD exacerbation were infrequently admitted to the ICU and withdrawal of treatment was the most common cause of death in these individuals after admission.²⁸ However, the data shows that survival following mechanical ventilation is actually better when no causative factor is identified for an exacerbation.²⁹ Thus, a COPD patient who presents with isolated respiratory failure cannot be “written off” as a lost cause. Short-term survival following an episode of mechanical ventilation for COPD ranges from 63% to 86%.^30,31,32 In contrast, long-range survival rates are worse—52%, 42%, and 37% at 1, 2, and 3 years.²⁸ Determining who will benefit from critical care utilization is difficult. Though medical comorbidities, prolonged (> 72 hours) mechanical ventilation and failure postextubation predict poor outcome,³¹ national guidelines do not currently support the use of clinical scoring systems for management of COPD exacerbations.³³ Thus, physicians have little to guide them when judging the appropriateness of critical care intervention in this population.

While many patients make excellent recoveries, others that survive COPD exacerbations suffer with chronic critical illness that impairs their quality of life and causes tremendous financial hardships for their families. Overall, it is estimated that between 5% and 10% of patients requiring mechanical ventilation for acute respiratory failure will develop chronic critical illness.^34,35,36 The cost of caring for these patients is estimated to be over 20 billion in the United States and will likely rise as the population continues to age.³⁷ Delirium is a serious adverse event that complicates the management of ICU patients and increases their morbidity and mortality.³⁸ The diagnosis of COPD has been identified as an independent risk factor for delirium in cardiothoracic ICU patients.³⁹ In addition, COPD has been reported to increase the risk of developing ARDS in ICU patients.^40,41 The specific mechanisms responsible for this increased susceptibility have yet to be determined. Clearly, an enhanced understanding of the underlying disease pathogenesis is needed for improved prognostication and to develop better means of treating this disease and preventing its associated complications in the ICU.

COPD research funding has been disproportionately low given its impact on public health.⁴² This is undoubtedly due to the fact that this is perceived as a “self-inflicted” disease. For the most part, if people did not smoke they would not develop the disease and if they stopped smoking the disease, if present, would not progress as quickly. This naturally raises the question why should scarce public research dollars be used to investigate a disease that has such a clear-cut solution? However, this perspective clearly ignores the history of this product over the past century. Cigarettes are not marketed toward 20, 30, or 40 years olds.⁴³ Rather, cigarettes, whether in print, sporting events or the movies are aggressively pitched towards adolescents.⁴⁴ This is a group that is more susceptible to peer pressure and influence and less able to gauge the true risks of this habit. In fact, billions of dollars are spent annually marketing this product to young people worldwide.⁴⁵ Unfortunately, adolescents and the public as a whole have a very poor understanding of the addictive potential of this product. Cigarettes are not ground up tobacco leaves wrapped in paper. They are a carefully engineered product laced with chemicals, such as ammonia that speed the absorption of nicotine in the body.⁴⁶ Nicotine reaches the brain within seconds of a puff on a cigarette creating a sensation of euphoria.⁴⁷ These rapid effects are powerfully reinforcing psychologically and help to explain why smoking cessation success rates are so poor.⁴⁸ There are 17 nicotinic acetylcholine receptor subunits in humans that trigger multiple responses.⁴⁹ Cigarettes are a legal product and tobacco companies are permitted to spend billions of dollars to get people to try their product despite the potential of addiction and reliance on this product. Given this current state of affairs, those who suffer from COPD are as deserving of effective treatments as those who suffer from other lifestyle influenced conditions such as diabetes, hypertension, and heart disease. Equally, there are numerous clinical manifestations that occur with second hand smoking, with individuals who involuntarily undergoing smoke exposure. Thus, there is an urgent need to better understand the pathogenic processes that cause this disease so that more effective therapies can be developed.

While we know that cigarette smoke exposure will induce pathologic effects in the lungs of smokers, the mechanisms by which cigarette smoke mediates these changes remain incompletely understood. Studies in humans frequently utilize biological materials from patients who are already in the advanced stages of the disease. Although helpful, these studies may not provide insights into the processes by which cigarette smoke promotes the development of the disease state in the lung. Performing prospective studies in humans could address these limitations; however, such studies would be time-consuming and expensive as COPD is an insidious disease that evolves over many years.⁵⁰ Over the long term, it would be difficult to perform these analyses in a rigorous scientific manner that would limit the confounding effects of other dietary, genetic and environmental variables. Moreover, obtaining biological specimens over this length of time would be costly and require years to answer scientific questions. For this reason, animal models of COPD are needed to better understand the underlying disease mechanisms. After Laurell and Erickson's observation that A1AT (alpha-1-anti-trypsin) deficiency resulted in the premature development of emphysema,^51,52 the role of elastase in this disease became center focus. A1AT protein is an abundant circulating anti-protease that binds and neutralizes neutrophil elastase within the lung.⁵³ Thus, it was postulated that the unopposed action of elastase would degrade elastin, which is abundant in the alveolar wall, thereby causing the destruction of lung tissue.⁵⁴ This theory was further supported by the finding that intratracheal elastase induced the development of emphysema in rats.⁵⁵ This was one of the first animal models of the disease and it had a tremendous influence on the research direction of the pulmonary field for the subsequent 30 years. After this discovery, emphysema was primarily regarded as a disease of elastin degradation that resulted from an elastase/anti-elastase imbalance in the lung. Although this model provided important insights into the disease, it had numerous shortcomings that limited its applicability to the human disease. For one, emphysema in this animal model develops within 3 weeks of exposure.⁵⁶ This rapid onset of development does not mimic the human disease, which often takes decades to develop. Thus, it is likely that distinct biological processes are responsible for the pathologic changes that are required for disease initiation and progression to occur. Secondly, by its design, the elastase animal model cannot address the impact of cigarette smoke exposure on the lung. Although cigarette smoke induces expression, release and activation of lung elastases,^57,58 it also triggers a myriad of other effects that will not be replicated by an elastase disease model. Thirdly, the elastase model reproduces the alveolar destruction that occurs in emphysema but it does not replicate other disease features such as mucus plugging, bronchiolitis and altered lung inflammation.⁵⁹ Indeed, the hallmark features of human COPD such as chronic lung inflammation, impaired lung function, emphysema, mucus hypersecretion, vascular injury, and small airway remodeling are not well represented in this model. Thus, researchers have sought to utilize smoke exposure models to gain more relevant insights into the disease.

Developing a smoke exposure model that would generate emphysematous changes was a laborious process. The early attempts to develop smoke exposure models were complicated by the long exposure time required to develop the disease and the variable effects of cigarette smoke in the exposed animals. Indeed, some of the first studies reported cellular proliferation and mucus metaplasia but not the classical alveolar destruction seen in the human disease.^60,61,62 Hautamaki et al were the first group to successfully overcome these challenges in a mouse exposure model.⁶³ In their model, they used a pump to circulate the cigarette smoke generated by the burning of 2 cigarettes to a Plexiglas chamber containing the mice. Carbon monoxide (CO) measurements showed that the mice had CO levels that were comparable to human smokers. By exposing mice in this manner daily for several months they successfully generated alveolar destruction within the lungs of the exposed mice. Moreover, they demonstrated that this destruction was dependent on the expression of an elastase, matrix metalloproteinase-12 (MMP-12). Establishing an emphysema model that successfully generated alveolar destruction was one of the most important accomplishments in the pulmonary field in the past 2 decades. Today, investigators are actively using variations of this model to obtain important new insights into the mechanisms of this disease.

The 2 most common smoke exposure models that have been utilized are the nose only and the whole body exposure apparatus.

The nose only system requires restraining the mouse so that their nose is inserted into a cone where they inhale the cigarette smoke.⁶⁴ This generates a uniform exposure that produces emphysematous changes. However, the prolonged periods of restraint are stressful for the mice and the machine can usually accommodate only a limited number of mice (eg, the Jaeger system has 18 ports which is depicted in Figure 57A–1A). In contrast, whole body exposure systems expose mice to a mixture of both passive and mainstream smoke released from a burning cigarette and mainstream smoke, which is actually smoke aspirated through the cigarette using a pump.⁶⁵ The passive and mainstream smoke streams are mixed and then propelled by a fan to a chamber containing the mice that are housed within their cages. The advantage of this system is that the mice freely move about and have access to food and water (Figure 57A–1B). Thus, mice in this system can be exposed for longer periods of time. In addition, the whole body exposure system allows for the exposure of large groups of mice. Some systems allow the exposure of up to 120 mice at a time enabling researchers to use large numbers of mice and to perform multiple experiments simultaneously. An important note about whole body exposure systems is that the exposure intensity needs to be monitored very carefully. Cigarette smoke is removed from cages via exhaust valves that are typically maintained with a small aperture in order to allow smoke levels to build up within the chambers. Cigarette smoke releases a large amount of tar that can clog these valves and minimize the flow of cigarette smoke, which can allow for smoke levels to build up to toxic levels. If the tar obstruction of the valve becomes too great, all smoke/air flow stops and the mice receive no smoke exposure. Both of these outcomes can ruin a carefully planned experiment. Thus, the mice have to be closely monitored to ensure that airflow is circulating properly and that toxic levels of cigarette smoke are not building up.

This is accomplished by measuring total particulate matter (TPM) concentration within the chamber and carbon monoxide levels in the mice. Typically, the TPM is maintained at 80 to 100 mg/m³, which will produce CO levels in the mice of 10% to 12%.⁶⁵ These levels are well tolerated by the mice and produce the alveolar lung tissue destruction.^63,65 TPM can be monitored using a filter sample unit fitted with a diaphragm pump and a timer. Utilizing sampling air to measure particulate matter in a specific volume sampled over time allows accurate TPM measurements and consistent exposure to animals.

Although many animals have been used in COPD studies, mice offer very clear advantages, which have lead to their becoming the dominant animal model for this disease. For one, mice are small in size and the costs of feeding and housing these animals are far less than dogs, sheep, and large rodents. Also, the mouse genome has been extensively characterized and there is a plethora of antibody, molecular probes and equipment modified for mouse anatomy available for studies in these animals. Likewise, it is far more cost-effective to generate genetically manipulated mice than it is to do so in other species. The gestational period for a mouse is 21 days compared to a time period of up to 74 days in guinea pigs. This allows investigators to generate large numbers of genetically altered mice in a relatively short period of time. Exposing mice to smoke for 1 year represents approximately 50% of the animal's lifetime, thereby allowing a better representation of lifetime smoke exposure. These cost advantages allow for large numbers of animals to be utilized for smoke exposure studies. This is extremely important when studying this disease, as the effect of cigarette smoke on the lung is highly variable. In humans, some estimates state that only 15% of smokers will develop emphysematous changes in the lung⁶⁶ and the susceptibility of mice to cigarette smoke is also quite variable.^65,67 Thus, exposing large numbers of mice increases the power of a study to detect significant differences in emphysema between control and genetically altered mice. The typical enlargement of airways observed after 6 months of smoke exposure is depicted in Figure 57A–2.

Mice provide the best opportunity to investigate the mechanisms of this disease. However, as noted previously, not all strains of mice are equally responsive to cigarette smoke.^65,67 Some strains of mice, such as C57Bl/6J, are relatively resistant to cigarette smoke and develop increases in their mean linear intercept (MLI), a parameter of airspace enlargement, which range from 15% to 20% after 6 to 12 months of exposure.⁶⁷ Because of this, smoke exposure studies using C57Bl/6J mice require large numbers of mice to be exposed for long periods of time. This greatly increases the time and expense needed to carry out these exposure studies. In addition, the C57Bl/6J mice do not develop increased lung compliance following chronic cigarette smoke exposure.⁶⁵ Numerous mouse strains have been utilized for smoke exposure studies. However, A/J mice are the most sensitive to cigarette smoke and develop increases in their MLI in the range of 20% to 30% after only 2 months of exposure,⁶⁵ as depicted in Figure 57A–3.

Because of this, the A/J mouse is becoming the preferred strain for smoke exposure studies in mice.⁶⁸ Unfortunately, many genetic mouse models are developed in a C57Bl/6J background.

Crossing these mice into a more sensitive A/J background requires several generations of backcrossing. This is a costly and time-consuming endeavor. If the primary endpoint of one's research is emphysema, it may be worthwhile to cross into an A/J background. These mice need less smoke exposure time and develop greater emphysema potentially making it easier to detect changes between groups. On the other hand, if one's focus is on inflammation, apoptosis, and protease expression then there is no evidence that A/J mice are superior to C57Bl/6J for examining these parameters.

Inflammation

The smoke exposure model has provided important new insights into the role of inflammation in the development of emphysema. As observed in humans, cigarette smoke induces a pronounced airway and lung parenchymal inflammatory response, particularly in dendritic cells.^65,69 Indeed, smoke studies in mice demonstrated that monocyte chemoattractants, such as granulocyte monocyte chemoattractant factor (GM-CSF), endothelial cell monocyte activating protein (EMAPII) and monocyte chemoattractant protein (MCP-1) play a pivotal role in the development of smoke-induced inflammation and emphysema.^63,70,71 In fact, smoke mediates this inflammatory response by stimulating receptors for advance glycation end-products (RAGE) on the surface of alveolar macrophages.⁷² The importance of this cell type was underscored by the fact that deficiencies in macrophage number or function ameliorated the inflammatory and destructive changes to smoke exposure in mice.^63,73

In addition to macrophages, the murine smoke exposure model has also established that cigarette smoke stimulates the infiltration of CD4+ and CD8+ cells in the lung tissue.⁷⁴ This is significant, as T cells have been linked to emphysema development in the human disease.^75,76,77 In fact, chronic smoke exposure in mice generates an oligoclonal expansion of pathogenic CD4+ and CD8+ cells in a mouse model of COPD.⁷⁸ Furthermore, smoke exposure in mice induced IFN-γ expression by NK cells in the lung.⁷⁹ By stimulating IFN-γ, these cells promote a Th1 inflammatory response that causes lung remodeling and tissue destruction.⁸⁰ More recently, the smoke exposure model identified that IL-17 production by Th17 cells plays an important role in the pathogenesis of this disease.^81,82 These Th17 cells from smoke exposed mice were capable of generating emphysematous changes when transferred into normal recipient mice.⁸³ While T cells play a central role in the disease pathogenesis, studies in humans show that lymphoid follicles containing B cells are associated with emphysema.^84,85 However, the role of B cells in the development of the disease has been controversial.⁸⁶ The smoke exposure model, however, has provided key new insights into the role of these cells in this disease. In mice, the smoke-mediated increase in B-lymphocytes correlated with the development of air space enlargement⁸⁷ and neutralizing the B-cell attracting chemokine CXCL13 prevented smoke-induced emphysema.⁸⁸ Thus, data from the mouse smoke exposure model suggests that both B and T cells are participating in the evolution of this disease.

While the smoke exposure model has identified new roles for T and B cells in COPD, it has confirmed and expanded our understanding of the effect of neutrophils in this disease. Smoke exposure studies in mice affirmed that proteases released from neutrophils break down matrix elements in the lung^89,90 and inhibiting these proteases exerts a protective effect in this disease.⁹¹ In addition, they demonstrated that cigarette smoke and nicotine sustain inflammatory responses by blocking the spontaneous death of lung neutrophils.⁹² Furthermore, the smoke exposure model showed that IL-1α is central to the initiation of smoke-induced neutrophilia.⁹³ However, though blocking IL-1R1 prior to smoke exposure prevents the influx of neutrophils and the development of emphysema,⁹⁴ blocking IL-1 late in the disease course could exacerbate lung injury by preventing the resolution of lung neutrophilia. Stimulation of IL-1R1 induces miR135b, which, in turn, suppresses lung inflammation by down regulating IL-R1 expression in the lung.⁹⁵ Thus, the use of IL-1 antagonists as a treatment for COPD will have to be approached with caution as inhibiting IL-1 in the late stages of the disease may accentuate lung tissue injury by heightening and sustaining the influx of neutrophils in the lung.

Animal models have shown that cigarette smoke triggers the production of damaging proteases in the lung.^96,97 These proteases augment lung inflammation by degrading key structural elements that release chemotactic peptides in lung.⁹⁸ Studies in mice show that anti-proteases, such as alpha one antitrypsin, antagonize these effects to prevent the development of smoke-induced emphysema.^99,100 Specifically, A1AT blocks the production of MMP-12 and TNF-α in alveolar macrophages from mice¹⁰¹ and prevents caspase-3 activity and apoptosis in lung endothelial cells.¹⁰² Animal studies from the mouse smoke exposure model show that both MMP-12 and TNF-α play a central role in the development of smoke-induced emphysema.^63,103 Likewise, endothelial cell death is a key process in the onset and progression of COPD.^104,105 Thus, A1AT, by inhibiting these responses, counteracted key processes responsible for this disease. Alteration on lung signaling can have a profound effect on the protease/antiprotease balance in the lung. Studies in mice show that cigarette smoke inactivates the histone deacetylase SIRT1 leading to the up regulation of MMP-9 and downregulation of TIMP-1.¹⁰⁶ This proteolytic imbalance resulted in the development of smoke-induced emphysema in mice.¹⁰⁷ Furthermore activation of the tyrosine kinase c-Src induced the expression of MMP-9 and MMP-12 both in lung epithelial cells and the lungs from smoke exposed mice.¹⁰⁸ These findings indicate that targeting c-Src may counter the proteolytic process that lead to lung inflammation and tissue destruction in this disease.

Oxidants

The use of animal models has greatly enhanced our understanding of the role of oxidants in the development of emphysema. Chronic smoke exposure studies document the accumulation of oxidative injury in the lungs of exposed mice.^109,110 Importantly, oxidants can inactivate A1AT thereby negating a key protective mechanism in the lung.¹¹¹ In fact, oxidized A1AT actually enhances the release of MCP-1 from lung epithelial cells. Although cigarette smoke exposure is associated with oxidative stress and the induction of injury lung responses,¹¹² direct evidence of the role of smoke-derived oxidants in the pathogenesis of the disease had been lacking. Studies in nuclear factor (erythroid-derived 2)-like 2 (Nrf2) deficient mice demonstrated that lung antioxidant expression was a key determinant of smoke-induced inflammation and tissue destruction. Nrf2 is a transcription factor that is normally bound to its inhibitor Keap1 in the cytosol. In response to oxidative stress, Keap1 dissociates from Nrf2 allowing it to translocate to the nucleus where it turns on the expression of lung antioxidant genes. Thus, Nrf2 knockout mice had deficient antioxidant responses and this resulted in greater oxidative stress, lung inflammation and airspace enlargement.¹¹³ In contrast, Keap1 knockout mice had decreased inflammation and oxidative injury in response to acute smoke exposure.¹¹⁴ Furthermore, it was demonstrated in mice that enhancing lung antioxidant expression effectively counteracts the inflammation and proteolytic responses to acute and chronic cigarette smoke exposure. Indeed, mice with the transgenic expression of the antioxidant superoxide dismutase-1 (SOD1) were completely protected against smoke-induced inflammation, protease expression, oxidative injury and lung tissue destruction.⁵⁶ This was the first direct demonstration that countering smoke-induced oxidants could have a beneficial impact in this disease. Subsequently, it has been shown the extracellular SOD (EC-SOD) similarly protects against smoke-induced emphysema in mice by preventing the oxidative fragmentation of the extracellular matrix.¹¹⁵ In addition, the antioxidants glutathione peroxidase-1^116,117 and thioredoxin exert similar protective effects in smoke exposed mice.^118,119 These studies offer hope that enhancing the antioxidant defenses of the lung can prevent the damaging effects of chronic smoke exposure in the lung. Current antioxidant approaches in humans are limited by pharmacokinetic factors that restrict the lung bioavailability of exogenously administered antioxidants.¹²⁰ Indeed, the n-acetyl cysteine dose used in the BRONCUS study¹²¹ was shown not to significantly enhance lung antioxidant defenses in humans.¹²² This may explain the disappointing results from this clinical trial. Lastly, though these studies indicate that oxidants are contributing to the injurious effects of cigarette smoke exposure, the genetic ablation of the NADPH oxidase actually enhances airspace enlargement and inflammation in smoke exposed mice.¹²³ Free radicals are key mediators of intracellular signaling events. Thus, the complete absence of lung oxidases may alter the ability to the cell to respond to the damaging effects of cigarette smoke exposure in the lung. Rather than an indiscriminate antioxidant approach it may be more effective to identify redox-regulated processes that could be targeted with specific pharmacotherapies.

Apoptosis

Apoptosis is a critical event in the development of lung tissue destruction and remodeling in COPD.¹²⁴ However, it is difficult to determine how cigarette smoke induces lung apoptosis using only clinical specimens. Thus, the animal exposure model provides a powerful tool for better understanding the apoptotic mechanisms that are triggered by cigarette smoke. Studies in mice showed that apoptotic responses occur in the lung even after short-term cigarette smoke exposure.¹²⁵ Furthermore, these studies show that antioxidant supplementation blocks apoptosis in response to cigarette smoke in these mice. The importance of redox factors on lung apoptosis was further demonstrated in Nrf2 knockout mice. The loss of antioxidant induction in Nrf2 knockouts accentuated the development of apoptosis in both lung epithelial and endothelial cells.¹¹³ Thus, these smoke exposure studies have helped to identify how redox biology influences cell fate in resident lung cells exposed to cigarette smoke.

As observed with oxidants, studies in mice have shown that cytokines, such as IFN-γ, also play a critical role in smoke-induced apoptosis in the lung. Indeed, cigarette smoke up regulates IFN-γ, which then acts through its receptor CCR5 to trigger apoptosis and lung tissue remodeling.⁸⁰ Similarly, TNF signaling has been shown to play an important role in the induction of apoptosis in mouse smoke exposure models. Mice that lacked expression of TNF-receptor I or TNF-receptor II (TNFR1 or TNFR2) were protected against the development of smoke-induced apoptosis, particularly within type II pneumocytes.¹²⁶ This protection was associated with decreased lung inflammation and the preservation of normal lung architecture. Cigarette smoke also induces IL-6 and this cytokine-triggered alveolar apoptosis and emphysema in smoke exposed mice.¹²⁷ In addition to epithelial cells, smoke studies in mice found that IL-18 and the chemokine receptor CXCR3 were important mediators of endothelial cell apoptosis and lung injury.^128,129 Over the past 10 years, there has been an increasing awareness of the role of the lung endothelium in the pathogenesis of COPD.⁵⁹ Vascular endothelial growth factor (VEGF) maintains endothelial integrity and prevents cell death.¹³⁰ Impairing VEGF signaling by inhibiting its receptor VEGFR2 augmented inflammation and endothelial dysfunction in smoke exposed mice.¹³¹ This is important as cigarette smoke induces oxidative stress that blocks VEGFR2 activation and impairs VEGF signaling within the endothelium.¹³² Studies in both mice and humans show decreased expression of VEGF within the airways in response to cigarette smoke exposure.¹³³ Moreover, studies in mice show that endothelial VEGF expression is similarly decreased in response to cigarette smoke exposure.¹³⁴ Together, these findings establish that altered VEGF signaling within the lung endothelium causes endothelial cell death and tissue destruction in this disease. Future studies are ongoing to determine whether antagonizing these effects can preserve endothelial integrity and ameliorate the inflammatory and destructive changes that occur in this disease.

As noted in the prior section, the cigarette smoke exposure model has provided key insights into the mechanisms by which cigarette triggers lung inflammation, protease expression, apoptosis, and tissue destruction. These studies have enabled us to better understand why cigarette smoke causes lung disease. Much less attention, however, has been paid to determining how the lung recovers from the damaging effects of cigarette smoke. This is an important question as enhancing recovery mechanisms may be a means of countering or reversing the harmful biological responses that occur in this disease. Several recent reports have begun to address this issue and have laid the groundwork for future study.^135,136

It is well established that FoxP3+ T regulatory cells mediate the resolution of inflammatory responses in the lung. In an LPS lung injury model, depletion of these cells exacerbated lung inflammation while adoptive transfer of these cells, even 24 hours after LPS challenge, accelerated lung recovery.¹³⁷ In smoke exposed mice, there is an accumulation of T regulatory cells in the lung.¹³⁸ This suggests that these cells are functioning to limit the inflammatory responses to this stimulus in the lung. Unfortunately, with chronic exposure T cells were skewed to a Th17 phenotype and away from the development of T regulatory cells.¹³⁹ While these studies point to a protective role for T regulatory cells in COPD, a recent study in mice found that in utero smoke exposure increased T regulatory activity, which impaired tumor clearance later in life by impeding the function of cytotoxic T cells.¹⁴⁰ Given these conflicting findings, further studies are needed to determine whether augmenting T regulatory cell activity will have a beneficial or adverse effect in this disease.

The clearance of inflammation is not a passive process but rather involves innate resolution mechanisms that are activated at the initiation of injury. In the lung and other organs, a class of natural lipid-derived mediators (lipoxins, resolvins, protectins, maresins) is produced to resolve inflammation in a manner that does not suppress the immune system. Lipoxins are derived from cell membrane arachidonic acid and suppress inflammation in asthma¹⁴¹ and lung injury models.¹⁴² Serum amyloid A (SAA) protein, which blocks the effects of lipoxins, is increased in COPD and administering SAA enhances neutrophilic responses in mice.¹⁴³ Thus, the loss of lipoxin activity may contribute to neutrophilic inflammation in this disease. Resolvins, protectins, and maresins are derivatives of omega-3 polyunsaturated fatty acids (ω-3-PUFA). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are 2 essential ω-3-PUFAs that are converted into these bioactive lipid mediators in order to regulate the local resolution of inflammation.¹⁴⁴ Animal models have demonstrated that resolvins promote the resolution of lung inflammation due to bacterial pneumonia,¹⁴⁵ LPS,¹⁴⁶ or asthma.¹⁴⁷ Resolvins exerted these effects by blocking mast cell degranulation,¹⁴⁸ promoting neutrophil clearance¹⁴⁹ and suppressing NF-κB activation.¹⁴⁶ Using the mouse smoke exposure model it was shown that resolvin D1 decreases neutrophilic inflammation and up regulates the anti-inflammatory cytokine IL-10.¹³⁵ Given these effects, further studies in this model are needed to determine whether resolvins prevent inflammation and tissue destruction in response to chronic smoke exposure.

The cell contains a tightly coordinated network of kinases and phosphatases that switch proteins from the phosphorylated to the dephosphorylated state in order to cope with various physiologic challenges.¹⁵⁰ Cigarette smoke activates kinases that promote lung disease by stimulating inflammation,^108,151 up regulating proteases¹⁵² and inducing apoptosis.¹⁵³ While a significant body of research has elucidated the role that protein kinases exert in these processes,¹⁵⁴ much less is know about the effects of protein phosphatases. Protein phosphatase 2A is the primary serine threonine phosphatase of eukaryotic cells. Cigarette smoke activates PP2A both in human lung epithelial cells and mouse lung.¹⁵⁵ This activation occurs rapidly and limits the intensity and duration of smoke induced inflammation. Indeed, inhibiting PP2A in mice sustained and exacerbated the effects of acute smoke exposure.¹⁵⁵ Though acute smoke exposure activates PP2A, this response is lost with chronic smoke exposure potentially rendering the lung more susceptible to smoke-induced injury.¹⁵⁵ It is important to note that studies from the mouse smoke exposure model showed that PP2A protects against smoke-induced emphysema. In fact, the antioxidant glutathione peroxidase-1 (GPx-1) acted via PP2A to prevent smoke-induced inflammation and lung tissue destruction¹¹⁷ in mice. PP2A plays a pivotal role in preventing and resolving smoke-induced lung inflammation and injury. Thus, targeting PP2A activity in the lung may be an effective means of preventing the onset and progression of COPD.

As noted previously, COPD is a leading cause of death worldwide. Despite its importance, there are few specific therapies for this disease and only oxygen has been shown to impact on disease mortality. The lack of effective treatments is due in large measure to the limited knowledge of the underlying disease mechanisms. Over the past 20 years, the cigarette smoke exposure model in mice has provided valuable insights into the biological processes responsible for the disease pathogenesis. Today, the role of proteases, inflammation and apoptosis in the disease development is much better understood. In addition, researchers have identified key counter inflammatory mechanisms that serve to protect the lung against the damaging effects of cigarette smoke exposure. The knowledge gained from these studies has the potential to translate into effective treatments to prevent or reverse the course of this disease. Additionally, well-established smoke exposure models are now available to test potential new therapies prior to human clinical trials. However, the translational impact of these studies has yet to be realized for a multitude of reasons. For one, COPD is a complex disease that develops over years. Interventions administered during the early or pre stage of disease may not be effective when given to late stage patients. This is a daunting problem for COPD as this is a woefully under diagnosed disease and most cases are not detected till the disease is well established.¹⁵⁶ Unfortunately, most studies that utilize the mouse smoke exposure model administer the intervention before smoke exposure begins or before lung damage is established.¹⁵⁷ This is obviously not what happens with a patient who is newly diagnosed with the disease. Moreover, inhibiting some processes during the early period of exposure may prevent disease but blocking the same pathway in late stage disease may hinder needed compensatory responses. Therefore, the scientific community may be studying the prevention of disease initiation rather than recovery. This may be the case with TNF-α antagonists, which prevented COPD in animal models^158,159 but were ineffective and potentially harmful in clinical studies in humans.^160,161 In the future, animal exposure studies will need to be conducted after the disease is already established. The A/J strain of mice will be well suited for these studies because they develop significant emphysema after only 2 months of smoke exposure. Thus, the intervention could be begun at this time point to assess the potential benefit of an intervention. Though changes in study design will help, the limitations of the murine smoke exposure model have to be recognized. Most obviously, mice are not humans. They have shorter lifespans, less submucosal glands and more airway macrophages than their human counterparts.^162,163 Given these and other biological differences, the mouse model may not fully replicate what occurs in the human lung. Carefully conducted correlative studies in humans are, therefore, needed to confirm promising results obtained with this model. The statistician George E.P. Box once wrote, “All models are wrong, but some models are useful.”¹⁶⁴ The smoke exposure model, by definition, is an approximation of what occurs in the human lung. It can provide useful insights, but if used without discretion it can obfuscate the truth. The challenge for researchers moving forward will be to conduct exposure studies that integrate physiologic, biologic, and architectural endpoints and then to validate these findings with research using complementary in vitro and in vivo models and studies using human samples. Rushing to drug development or testing based on the results from the smoke exposure model alone is shortsighted especially considering the model's limitations. Nevertheless, if used wisely, this model can be an important component of a multifaceted research approach for this disease.