In the present study, we demonstrate an attenuating effect of obesity on LPS-induced lung injury and neutrophil trafficking in genetically hyperphagic (db/db) and diet-induced mouse models of obesity and implicate obesity-related defects in neutrophil chemotaxis in this diminished response. This is consistent with our previously published findings on obesity’s dampening effects on the inflammatory response in human ALI.
Previous work has suggested that obesity may have an attenuating effect on hyperoxic and ozone-induced lung injury models, although in the case of ozone exposure findings are mixed and appear to vary with the acuity of exposure and possibly the timing of examination (22
). These findings, in light of the early evidence that obesity may have a protective effect in human ALI/ARDS (7
), suggest that a clinically relevant alteration in the acute pulmonary inflammatory response may be associated with weight gain. Although limited work has explored this effect in animal models, leptin resistance (20
) and alterations in IL-6 signaling (22
) have been implicated in the attenuation of acute lung inflammation, but the possible role of obesity-associated neutrophil function defects has not previously been investigated.
Examining an LPS-induced lung injury model, we find that db/db and DIO animals show decreased airspace neutrophilia and attenuated capillary leak. Elevated levels of circulating neutrophils are seen in the obese mice in our studies, similar to findings we have previously reported in obese patients with ARDS (19
). This suggests that neutrophil mobilization in response to injury is not impeded in obesity, implicating a defect in the recruitment of blood-borne neutrophils to the airspace as the cause of attenuated injury and neutrophilia. Although such a finding could result from an abnormal pulmonary cytokine response, in our models the proinflammatory cytokine response appears to be normal. Furthermore, in the case of the db/db model of obesity, airspace neutrophilia is blunted even in this early phase of recruitment, suggesting that defects in neutrophil response may exist in obese animals, leading to impaired neutrophil migration into the lung. This is further suggested by our adoptive transfer studies in which neutrophils from obese animals show significantly impaired airspace migration when infused into lung-injured, lean recipients. Thus, obesity appears to confer an intrinsically impaired neutrophil migratory response that is independent of additional host defects that may accompany obesity.
Neutrophil diapedesis into the lung is a complex process, requiring endovascular rolling, adhesion, and subsequent chemokine-directed tissue migration to the alveolar space (36
). Although defects in neutrophil chemotaxis may arise from alteration in multiple cellular processes, we find that impaired response to the CXC chemokine KC is evident in obese neutrophils during calcium flux, which is the earliest signaling event that initiates chemotaxis. Associated with this impairment, we find evidence of significantly decreased surface levels of the CXCR2 receptor on neutrophils from db/db obese mice and to a lesser degree in DIO mice. The cause of this reduction in CXCR2 is unclear. Although obesity is known to be accompanied by chronic, low-level systemic inflammation, which could lead to CXC cytokine-mediated reduction in neutrophil CXCR2 display, plasma inflammatory cytokine levels including CXCR2 ligands KC and MIP-2 are not significantly different between naive obese and lean mice in either model of obesity (Figures E8). Even in the case of db/db-derived neutrophils, unknown mechanisms other than reduced CXCR2 display must contribute to obesity-associated neutrophil chemotaxis defects, given the disproportionate magnitude of this defect in relation to the observed reduction in surface CXCR2.
The finding of an obesity-linked primary defect in neutrophil function adds to the growing list of obesity-associated defects suggested to contribute to the attenuated pulmonary inflammatory response, including leptin resistance and abnormalities in IL-6 signaling, and is notably similar to our previously published findings examining lean mouse models of dyslipidemia (37
), a condition known to accompany obesity. In our previous studies, lean mice with diet-induced hypercholesterolemia demonstrated a small but significant reduction in pulmonary neutrophilia 24 hours after exposure to nebulized LPS, which was associated with defects in neutrophil chemotaxis as well as decreased neutrophil surface levels of CXCR2 (37
). Although the effects of hypercholesterolemia on neutrophil trafficking were less substantial than those we report here in obese mice, such findings suggest that hypercholesterolemia, present in both models of obesity in this report (Figure E9), may contribute to the obesity-associated defects in neutrophil chemotaxis.
Although the current literature is inconclusive, there are suggestions that obesity may affect the recruitment of neutrophils to the lung differently from recruitment to other sites. For instance, peritoneal recruitment of neutrophils may be augmented in obesity in sterile peritonitis (38
). How this may be reconciled with our current finding of obesity-associated impairment of neutrophil chemotaxis is unclear. We have previously described a similar paradox in hypercholesterolemic mice (37
) in which we found isolated neutrophils to have similar defects in chemotaxis associated with increased recruitment to the inflamed peritoneum despite impaired recruitment to the lung using the same inflammatory agents (LPS, Klebsiella
infection). The etiology of this difference is unclear but may involve augmented cytokine response in the peritoneum compared with the lung.
Examination of inflammatory cytokine levels in our models demonstrates that, although the initial pulmonary cytokine response in obese animals appears normal, a reduction in plasma IL-6 levels is seen in more established injury (24 h). This finding is similar to our previous findings in patients with ALI in which plasma IL-6 was found to be decreased in obese patients. MCP-1 and to a lesser degree IL-6 are reduced in the airspace of obese animals with lung injury at 24 hours (Figure E9). It is unclear whether this occurrence reflects a downstream effect of attenuated neutrophil recruitment (because neutrophils are an important source of IL-6 and MCP-1 release after lung injury [39
]) or an evolving defect in the monocyte/macrophage or pulmonary epithelial response during the course of lung injury. In the case of IL-6, this decrease in alveolar cytokine release appears to mirror the defect seen in systemic cytokine response.
Differences are evident between our two mouse models of obesity. The db/db and diet-induced obesity models showed similar defects in neutrophil chemotaxis and comparable attenuations in airspace neutrophilia and capillary leak in the setting of established lung injury (24 h). However, these models manifest subtle differences in other aspects of the inflammatory response, possibly attributable to their disparate mechanisms and durations of obesity. Although db/db mice are primarily hyperphagic and rapidly develop obesity on normal chow within 4 to 6 weeks of birth, DIO mice develop obesity as a product of high fat chow over the course of 20 weeks. Thus, full manifestations of the metabolic syndrome, such as vascular activation and injury, are likely to be greater at baseline in the DIO model compared with the db/db model. Such endothelial activation may account for the normal to increased early neutrophil recruitment seen in injured obese DIO compared with lean mice () (as well as the relatively normal lung myeloperoxidase content) that occurs in this model despite the demonstrated obesity-associated defects in neutrophil chemotaxis.
Several other notable differences between obesity models exist. Mice with diet-induced obesity appear to have less pronounced alterations in neutrophil recruitment, calcium flux, and CXCR2 expression compared with the db/db model of obesity, whereas lean mice in the diet-induced model have a blunted response to LPS injury compared with lean heterozygous db mice. Several factors may account for these findings. Lean mice in the diet-induced model are significantly heavier than lean mice in the db/db model (22.2 ± 0.9 g versus 32.1 ± 0.9 g; P
< 0.0001), suggesting that differences in weight might affect BAL neutrophilia even in the “lean” groups. Diet composition, which differs substantially between models, also has been shown to alter the inflammatory response (40
) and may contribute to the differences seen between models. Although we do not find a significant correlation between airspace neutrophilia and mouse age in our lung injury model, age has been shown to impair neutrophil chemotaxis response in mice and humans, independent of weight, through unclear mechanisms (41
), and this may augment the defect in DIO neutrophils independent of CXCR2 expression levels.
The development of spontaneous diabetes in the db/db mouse model is well known, and although our experiments were designed to limit the development of frank diabetes in the animals by using mice on a nondiabetogenic background (B6) and examining them at an age before the typical onset of diabetes (46
), we cannot exclude the possibility that early diabetes may have influenced inflammatory response in the obese db/db mice. Thus, we might expect that the multiple differences between the db/db and DIO obesity models would alter how obesity-associated defects in neutrophil function and recruitment are expressed. Despite this, the shared phenotype of impaired pulmonary inflammatory response and neutrophil dysfunction in both models suggests that the obese state itself has an overarching effect on the pathogenesis of lung injury.
In summary, we show that obesity has an attenuating effect on LPS-induced lung injury and neutrophil trafficking in two mouse models of obesity. This occurs despite an apparently normal early pulmonary cytokine response and with elevated levels of circulating neutrophils. Further examination revealed that the witnessed attenuation on pulmonary neutrophilia in both models may in part be due to obesity-related abnormalities in neutrophil CXCR2 signaling with associated defects in neutrophil chemotaxis. Taken together, these results suggest that neutrophil dysfunction may play a prominent role in what appears to be a complex, multifactorial process underlying the attenuation of lung injury in obesity. Further studies are warranted to better characterize and dissect these obesity-related alterations in neutrophil function.