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Acute respiratory distress syndrome (ARDS) is characterized by an excessive pulmonary inflammatory response. Removal of excess cholesterol from the plasma membrane of inflammatory cells helps reduce their activation. The secreted apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells to the plasma lipoprotein HDL. Here, we find that AIBP was expressed in inflammatory cells in the human lung and was secreted into the bronchoalveolar space in mice subjected to inhalation of LPS. AIBP bound surfactant protein B and increased cholesterol efflux from alveolar macrophages to calfactant, a therapeutic surfactant formulation. In vitro, AIBP in the presence of surfactant reduced LPS-induced p65, ERK1/2 and p38 phosphorylation, and IL-6 secretion by alveolar macrophages. In vivo, inhalation of AIBP significantly reduced LPS-induced airspace neutrophilia, alveolar capillary leak, and secretion of IL-6. These results suggest that, similar to HDL in plasma, surfactant serves as a cholesterol acceptor in the lung. Furthermore, lung injury increases pulmonary AIBP expression, which likely serves to promote cholesterol efflux to surfactant and reduce inflammation.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

OBJECTIVES: One of the hallmarks of severe pneumonia and associated acute lung injury is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and acute lung injury. DESIGN: Controlled human and murine in vivo and ex vivo experimental studies. SETTING: Research laboratory of a university hospital. SUBJECTS: Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. INTERVENTIONS: Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated acute lung injury were immunostained for leptin. Human bronchoalveolar lavage samples obtained after lipopolysaccharide-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with Escherichia coli- or Klebsiella pneumoniae-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E. coli model. Bronchoalveolar lavage neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. MEASUREMENTS AND MAIN RESULTS: Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human bronchoalveolar lavage following lipopolysaccharide instillation. Bronchoalveolar lavage neutrophilia in uninjured and infected mice was increased and lung bacterial load decreased by airway leptin administration, whereas bronchoalveolar lavage neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by lipopolysaccharide, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated toward leptin in vitro, and this required intact signaling through the Janus Kinase 2/phosphatidylinositol-4,5-bisphosphate 3-kinase pathway. CONCLUSIONS: We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

RATIONALE: We have shown that obesity-associated attenuation of murine acute lung injury is driven, in part, by blunted neutrophil chemotaxis; yet, differences were noted between the two obesity models studied. We hypothesized that obesity-associated impairment of multiple neutrophil functions contributes to increased risk for respiratory infection, but that such impairments may vary between murine models of obesity. METHODS: We examined the most commonly used murine obesity models (diet-induced(DIO), db/db, CPEfat/fat, and ob/ob) using a K.pneumoniae pneumonia model and LPS-induced pneumonitis. Marrow-derived neutrophils from uninjured lean and obese mice were examined for in vitro functional responses. RESULTS: All obesity models showed impaired clearance of K. pneumoniae, but in differing temporal patterns. Failure to contain infection in obese mice was seen in the db/db model at both 24 and 48h, yet this defect was only evident at 24h in CPEfat/fat and ob/ob models, and at 48h in DIO. LPS-induced airspace neutrophilia was decreased in all models, and associated with blood neutropenia in the ob/ob model, but leukocytosis in the others. Obese mouse neutrophils from all models demonstrated impaired chemotaxis, whereas G-CSF-mediated survival, LPS-induced cytokine-transcription, and MAPK and STAT3 activation, were variably impaired across the four models. CONCLUSIONS: Obesity-associated impairment of host response to lung infection is characterized by defects in neutrophil recruitment and survival. Yet, critical differences exist between commonly used mouse models of obesity, and may reflect variable penetrance of elements of the metabolic syndrome and other factors.

We have previously reported that obesity attenuates pulmonary inflammation in both patients with acute respiratory distress syndrome (ARDS) and in mouse models of the disease. We hypothesized that obesity-associated hyperleptinemia, and not body mass per se, drives attenuation of the pulmonary inflammatory response and that this effect could also impair the host response to pneumonia. We examined the correlation between circulating leptin levels and risk, severity, and outcome of pneumonia in 2 patient cohorts (NHANES III and ARDSNet-ALVEOLI) and in mouse models of diet-induced obesity and lean hyperleptinemia. Plasma leptin levels in ambulatory subjects (NHANES) correlated positively with annual risk of respiratory infection independent of BMI. In patients with severe pneumonia resulting in ARDS (ARDSNet-ALVEOLI), plasma leptin levels were found to correlate positively with subsequent mortality. In obese mice with pneumonia, plasma leptin levels were associated with pneumonia severity, and in obese mice with sterile lung injury, leptin levels were inversely related to bronchoalveolar lavage neutrophilia, as well as to plasma IL-6 and G-CSF levels. These results were recapitulated in lean mice with experimentally induced hyperleptinemia. Our findings suggest that the association between obesity and elevated risk of pulmonary infection may be driven by hyperleptinemia.

Mounting evidence suggests that obesity and the metabolic syndrome have significant but often divergent effects on the innate immune system. These effects have been best established in monocytes and macrophages, particularly as a consequence of the hypercholesterolemic state. We have recently described defects in neutrophil function in the setting of both obesity and hypercholesterolemia, and hypothesized that exposure to elevated levels of lipoproteins, particularly LDL its oxidized forms, contributed to these defects. As a model of chronic cholesterol exposure, we examined functional responses of bone marrow neutrophils isolated from non-obese mice with diet-induced hypercholesterolemia compared to normal cholesterol controls. Chemotaxis, calcium flux, CD11b display, and F-actin polymerization were assayed in response to several chemoattractants, while neutrophil cytokine transcriptional response was determined to LPS. Following this, the acute effects of isolated LDL and its oxidized forms on normal neutrophils were assayed using the same functional assays. We found that neutrophils from non-obese hypercholesterolemic mice had blunted chemotaxis, altered calcium flux, and normal to augmented CD11b display with prolonged actin polymerization in response to stimuli. In response to acute exposure to lipoproteins, neutrophils showed chemotaxis to LDL which increased with the degree of LDL oxidation. Paradoxically, LDL oxidation yielded the opposite effect on LDL-induced CD11b display and actin polymerization, and both native and oxidized LDL were found to induce neutrophil transcription of the monocyte chemoattractant MCP-1. Together these findings suggest that chronic hypercholesterolemia impairs neutrophil functional responses, and these defects may be in part due to protracted signaling responses to LDL and its oxidized forms.