There are multiple pathways linking metabolism to immunity (6
), suggesting that obesity may influence immune function and affect the course of inflammatory diseases, such as asthma. Studies in humans and animals have supported a link between obesity and asthma (3
), demonstrating potential effects of obesity on airway inflammation (21
), AHR (7
), and remodeling (20
). However, the exact molecular mechanisms that explain this association are not fully known. Our data demonstrate that APN-deficiency, which mimics one component of the obese state, enhances allergic airway inflammation in a murine model of chronic asthma. Although APN deficiency did not seem to modulate airway fibrosis in this model, surprisingly, we observed a dramatic increase in pulmonary arterial muscularization associated with pulmonary hypertension. Obesity has been linked to pulmonary hypertension and pulmonary vascular remodeling, and it is now recognized that lung inflammation is an important component of the pathogenesis of some forms of pulmonary hypertension (34
). Thus, our data suggest that APN deficiency in obesity could also contribute to the development of pulmonary hypertension in the setting of inflammatory lung disease.
In normal individuals, APN circulates at very high levels (3–30 μg/ml), and is the most abundant adipose tissue–specific protein produced in humans (9
). However, individuals with obesity have low plasma APN levels, presumably due to feedback mechanisms. APN has a wide range of metabolic, antiinflammatory and antiproliferative activities (9
), suggesting that decreased APN levels may contribute to the increased inflammatory state in obesity. Consistent with this, APN−/−
mice are predisposed to diabetes, vascular disease, and ischemia–reperfusion injury (9
). Furthermore, APN seems to affect aspects of organ tissue remodeling and vascular SMC proliferation in disease (9
). It has been proposed that APN may be a “starvation signal” that works to limit energy expenditure. Thus, secretion of APN in times of limited nutrient reserve would suppress processes, such as protein production and cellular proliferation, to preserve cellular energy for other processes. It follows that, in APN-deficient states, such as obesity, inflammation and remodeling may be more robust due to a lack of this suppression.
Links between APN and lung disease are not well defined; however, a few recent studies have suggested that APN can influence the development of airway inflammation (21
). APN is normally found at high levels in airway lining fluid, and lung macrophages from APN−/−
mice have been shown to be more activated at baseline compared with wild-type mice (28
). In addition, all of the four known receptors for APN, AdipoR1, AdipoR2, T-cadherin, and calreticulin (51
), are expressed on multiple cell types in the lung, including macrophages, endothelial cells, and SMCs. Thus, the molecular machinery necessary for APN signaling exists in the lung and airways in multiple cell types.
We initially examined the effects of APN deficiency in a model of acute asthma, and found no differences in measures of airway inflammation or AHR between wild-type and APN−/−
mice. We next examined the effects of APN deficiency in a model of mild chronic allergic airway inflammation, reasoning that APN deficiency may have more of an impact over a long period of time, and that mild inflammation would be less likely to reduce APN levels in wild-type mice, as seen in the more intense acute model of asthma. In addition, a model of chronic asthma may be more relevant to obese patients with asthma. In this model, we did not see a decrease in APN levels in wild-type mice, and APN−/−
mice had an increase in airway inflammation associated with increased accumulation of eosinophils and monocyte/macrophages in the airways. Consistent with the increased cellular recruitment, there were increased levels of the chemokines, CCL11, CCL2, CCL7, and CCL12, in the lung, providing a possible mechanism for the increased airway inflammation observed in APN−/−
mice. Our data are consistent with recent human data, which revealed an inverse correlation between serum APN levels and CCL11 levels in patients with obesity (60
). Alveolar macrophages from APN−/−
mice have recently been shown to spontaneously produce more TNF-α than wild-type cells (28
), which may contribute to increased CCL11 production in the lung. However, we did not see increased spontaneous levels of CCL11 or CCL24 in PBS-challenged mice, though, the eotaxins require IL-4/-13 either alone or in combination with TNF-α to be up-regulated (42
CCL11 is produced by multiple cell types in the lung during allergic inflammation, including macrophages (41
). Previous data indicate that APN can influence immune responses through actions on NF-κB signaling in macrophages (63
), a cellular pathway that has been linked to the development of allergic inflammation and CCL11 production (48
). Furthermore, in vitro
studies on macrophages have demonstrated that APN inhibits NF-κB activation and the production of IFN-γ, CXCL10/IFN-γ–inducible 10-kD protein, and TNF-α (50
). Similar to this, we demonstrate a significant reduction in IL-4/TNF-α–induced CCL11 expression in bone marrow–derived macrophages after APN pretreatment. Overall, these data suggest that APN may modulate allergic inflammation in part via effects on CCL11 and reduced eosinophil recruitment.
AHR develops in response to airway inflammation in this model of chronic asthma (37
). We also saw a modest increase in AHR in both wild-type and APN−/−
mice, as measured by changes in airways resistance and dynamic compliance in response to methacholine. OVA-challenged APN−/−
mice had a greater change in compliance in response to methacholine than OVA-challenged wild-type mice, whereas airways resistance increased in response to methacholine to a similar degree in both genotypes. Prior studies have demonstrated that C57BL/6 mice are more likely to manifest AHR with changes in dynamic compliance rather than airways resistance, reflecting a propensity for small airway inflammation in this strain in asthma models (65
). Thus, our data might suggest that the greater change in dynamic compliance in APN−/−
mice could reflect an increase in small airway inflammation. However, PBS-challenged APN−/−
mice also have a greater decrease in compliance compared with PBS-challenged wild-type mice in response to methacholine. This suggests that there is a baseline decrease in dynamic compliance in mice with APN deficiency, which may also explain the differences between wild-type and APN−/−
mice after OVA challenge. Recent data suggest that naive APN−/−
mice develop spontaneous emphysema with macrophage activation (28
); thus, the decrease in dynamic lung compliance may be due to an increase in small airway closure from reduced lung elastic recoil and/or an increase in baseline airway reactivity. We did not see significant airspace enlargement in our APN−/−
mice; however, quantitative measurements of mean airspace chord length were not performed in our studies.
The most unexpected finding in our study was the dramatic increase in pulmonary arterial muscularization and pulmonary hypertension seen in the APN−/−
mice relative to wild-type mice in the model. In models of atherosclerosis, APN−/−
mice develop increased vascular SMC proliferation in systemic arteries (35
), but our study is the first to report similar changes in the pulmonary vasculature. Although asthma is not usually associated with the development of pulmonary vascular disease, inflammation is now recognized as an important stimulus for the development of pulmonary hypertension (30
). Interestingly, lung vascular remodeling has been reported in humans with asthma (in small vessels associated with inflamed airways) (67
), and more recently in murine models of allergic airway inflammation (30
). Male apolipoprotein E–deficient mice on a high-fat diet develop pulmonary hypertension associated with lower APN levels compared with wild-type mice (29
). Treatment of these mice with the peroxisome proliferator–activated receptor-γ activator, rosiglitazone, resulted in higher plasma APN levels and complete regression of pulmonary hypertension and pulmonary artery remodeling. Thus, these data suggest that APN may modulate PASMC proliferation and/or migration in response to inflammation or injury.
Eosinophils and macrophages are an important source of growth factors in allergic inflammation, and are essential for airway remodeling in asthma (53
). Thus, it is possible that the increased accumulation of these cells in APN−/−
lungs may contribute to the more intense pulmonary vascular remodeling found in APN−/−
mice via enhanced growth factor production. We did see an increase in the RNA levels of several growth factors important in remodeling and SMC proliferation (e.g., transforming growth factor-β, PDGF-α, PDGF-β, and plasminogen activator inhibitor-1) after OVA challenge in both wild-type and APN−/−
mice; however, there was no difference in the levels of these factors between the two genotypes. These differences do not exclude differences in protein levels, growth factor activation (or activity), or microanatomical differences (i.e., around blood vessels) of these factors. In addition, recent in vitro
studies have demonstrated that APN inhibits growth factor–mediated proliferation of murine PASMCs (29
). Growth factors stimulate SMC proliferation in part through effects on the mammalian target of rapamycin–S6 kinase pathway (75
), and APN has been shown to inhibit growth factor–mediated stimulation of mammalian target of rapamycin via AMPK activation (11
). Thus, the loss of direct suppressive effects of APN on PASMC proliferation could lead to the increase in pulmonary arterial muscularization found in APN−/−
mice, independent of differences in inflammation or growth factor activity.
If the increase in PASMC proliferation seen after OVA challenge resulted largely from direct effects on PASMC, we might expect to see similar changes in the vessels with other models of pulmonary vascular remodeling. However, in the hypoxia model of pulmonary hypertension, APN−/−
mice developed an amount of remodeling and pulmonary hypertension similar to that of wild-type mice. Although these data might suggest that the effects of APN deficiency on pulmonary vascular remodeling may be specific to allergic inflammation, recent studies have demonstrated that hypoxia suppresses APN secretion in wild-type mice (84
); thus, any differences between wild-type and APN−/−
mice may be attenuated.
In conclusion, our data suggest that low APN levels may increase asthmatic inflammation in obese individuals. We also observed the unexpected and novel finding of an increase in pulmonary arterial muscularization and pulmonary hypertension with APN deficiency in allergic inflammation, suggesting that coexisting inflammatory lung disease and obesity may predispose individuals to pulmonary hypertension due, in part, to reduced plasma APN levels.