The lung is not widely conceived of as a cholesterol-sensitive organ. However, cholesterol does play a pivotal role in physiological processes that are unique to the lung, including surfactant production by alveolar epithelial cells (22
) and regulation of the biophysical properties of surfactant (24
). Moreover, the importance of steady-state cholesterol flux to maintenance of normal lung homeostasis was recently revealed by reports of profound cholesterol overload of the lungs in Abcg1−/−
). As cholesterol overload potentiates innate immune and inflammatory responses of the macrophage (1
), and pharmacologic treatments reducing cellular cholesterol attenuate pulmonary innate immunity (4
), we predicted that the Abcg1−/−
lung would be primed for more robust host defense responses.
Herein, we report that Abcg1−/−
mice indeed display enhanced pulmonary responses to both inhaled LPS and K. pneumoniae
, characterized by increased airspace cytokine and chemokine levels and increased PMN recruitment. ABCG1, previously shown to be expressed in both alveolar macrophages and alveolar epithelial cells (8
), plays distinct roles in hematopoietic and nonhematopoietic (i.e., structural) pulmonary cells. Hematopoietic ABCG1 deficiency suffices to enhance pulmonary responses to K. pneumoniae
; nonhematopoietic ABCG1 deficiency is not sufficient, but further enhances responses in the setting of hematopoietic ABCG1 deficiency (). An underlying role for the alveolar macrophage in the Abcg1−/−
phenotype was confirmed, as Abcg1−/−
alveolar macrophages produced increased TNF-α when stimulated with LPS ex vivo
Of interest, indirect regulatory roles for ABCG1 in intercellular communications in the lung were also identified. Nonhematopoietic, but not hematopoietic, ABCG1 deficiency was sufficient to increase the number of alveolar macrophages during K. pneumoniae
infection. Hematopoietic, but not nonhematopoietic (e.g., epithelial), ABCG1 deficiency enhanced K. pneumoniae
–induced BALF expression of LIX (CXCL5), a PMN-active chemokine of exclusive epithelial origin in the lung (18
), perhaps through parallel increases in IL-17 (), a known inducer of LIX and PMN recruitment in the lung (26
). The normal chemotaxis that we observed in PMNs from Abcg1−/−
mice suggests that increased PMN recruitment is likely secondary to up-regulated chemokines in the ABCG1-deficient lung. Moreover, the relative deficiency of ABCG1 in WT PMNs, as compared with WT macrophages (), suggests the interesting opportunity for future studies aiming to contrast the relative role of this protein in PMN versus macrophage biology.
Increased PMN recruitment to and degranulation within the infected Abcg1−/−
lung led us to investigate whether clearance of K. pneumoniae
was also enhanced. PMNs are the critical cellular effector of host defense against extracellular bacteria, and extracellular MPO has bactericidal activity through catalyzing the formation of hypochlorous acid (20
mice indeed displayed enhanced compartmentalization and clearance of bacteria in the lung. Interestingly, regulation of innate immunity by ABCG1 in vivo
appears to be tissue selective, as Abcg1+/+
mice had equivalent responses to intraperitoneal LPS and intravenous K. pneumoniae
. Moreover, whereas Abcg1−/−
mice had enhanced basal recruitment of PMNs to the airspace and lung parenchyma, they displayed normal circulating PMN counts.
Alveolar macrophages from Abcg1−/−
mice displayed a cell-intrinsic enhanced innate immune response (), consistent with prior investigations of ABCG1-deficient macrophages (2
). However, it seems likely that the “nonnaive” basal state of the ABCG1-deficient lung, characterized by increased numbers of macrophages and neutrophils, and up-regulation of cytokines known to prime leukocyte host defense functions (e.g., degranulation), likely also contributes to poising the lung for enhanced host defense. As seen in other reported models, we speculate that the increased mortality in infected Abcg1−/−
mice () may stem from overexuberant lung inflammation (Figure E4). The increased BALF protein in Abcg1−/−
mice () also suggests that compromise of pulmonary microvascular integrity may contribute. In support of this premise, ABCG1 has recently been shown to promote endothelial function in systemic blood vessels (27
). Finally, future studies will need to discern whether surfactant in Abcg1−/−
mice, previously reported to have abnormal composition (8
), also has defective function.
Although others have demonstrated in vitro
that the TLR hyperresponses of ABCG1-deficient macrophages stem from their increased cholesterol (3
), we did not confirm this intermediate mechanistic link in vivo
in the present study. As macrophages deficient in ABCA1, another ABC family cholesterol transporter, accumulate levels of cholesterol that are similar to those of Abcg1−/−
macrophages, but produce significantly lower levels of cytokines (25
), factors other than simple cholesterol accumulation are likely to be involved. Subcellular distribution of cholesterol is likely important. For example, endoplasmic reticulum stress induced by free cholesterol overload is reported to result in nuclear factor–κB activation in macrophages (1
). On the other hand, TLR4 is up-regulated on the surface of cholesterol-laden Abcg1−/−
macrophages, but not Abca1−/−
macrophages, suggesting the importance of plasma membrane cholesterol. Of interest, as LPS itself down-regulates ABCG1 in human macrophages (28
), promoting cellular cholesterol loading, it is possible that disinhibition of the macrophage via ABCG1 down-regulation may even play a role in the LPS response of WT macrophages. Future studies will be necessary to better clarify how the spatial organization of intracellular cholesterol impacts the inflammatory state and maturation of macrophages, and how ABCG1 contributes to these events.
The observation that pulmonary innate immunity is modulated by ABCG1 may bear important clinical implications. Interestingly, the human lung disease pulmonary alveolar proteinosis has been linked to ABCG1 deficiency, likely stemming from neutralizing autoantibodies against granulocyte-macrophage colony–stimulating factor, a macrophage growth factor required for ABCG1 induction (9
). Pulmonary alveolar lipoproteinosis associated with macrophage infiltration has also been noted in Niemann-Pick disease, a lipid storage disorder characterized by macrophage cholesterol overload (30
). Taken together, these rare diseases suggest that more prevalent functional polymorphisms of ABCG1 may possibly also influence pulmonary inflammation and host defense in human subjects. At least two single-nucleotide polymorphisms (SNPs) have recently been investigated in preliminary studies in humans: G2457A, a SNP of uncertain functional significance, and −257T > G, a promoter SNP reported to reduce ABCG1 transcription and to associate with severity of coronary artery disease among Japanese men (32
As ABCG1 is a target gene of the nuclear receptor, liver X receptor (LXR), and endogenous LXR ligands synthesized in the lung are reportedly reduced in chronic lung disease in human subjects (34
), it is also possible that relative deficiency of ABCG1 may promote or sustain common chronic inflammatory lung diseases. Conversely, a recent report from our group that synthetic LXR agonists reduce pulmonary innate immune responses (5
) suggests that such agents may operate, at least in part, through ABCG1 up-regulation, and that ABCG1 may be a manipulable molecular target in lung disease. Of potential concern, statins, which are now undergoing study as potential therapeutics in human acute lung injury and chronic obstructive pulmonary disease, reportedly reduce ABCG1 expression in macrophages through depletion of endogenous LXR ligands (35
), an effect that could possibly mitigate their anti-inflammatory potential in the human lung.
In summary, we identify ABCG1 as a potential link between cholesterol homeostasis and host defense responses in the lung. We propose that ABCG1-mediated cholesterol homeostasis serves as an important negative regulator of leukocyte trafficking to the lung and pulmonary inflammatory responses to the environment. However, future investigations will be necessary to discern whether ABCG1 influences pulmonary inflammatory phenotypes through cholesterol-independent mechanisms, to better clarify the mechanisms by which cellular cholesterol regulates inflammatory functions in alveolar cells, and to determine whether pharmacological manipulation of ABCG1 and cholesterol in the lung may be used to improve respiratory health in human subjects.