GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS.
Alveolar macrophages (AMs) normally respond to lipopolysaccharide (LPS) by activating Toll-like receptor (TLR)-4 signaling, a mechanism critical to lung host defense against gram-negative bacteria such as Pseudomonas aeruginosa. Because granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient (GM(-/-)) mice are hyporesponsive to LPS, we evaluated the role of GM-CSF in TLR-4 signaling in AMs. Pulmonary TNF-alpha levels and neutrophil recruitment 4 h after intratracheal administration of Pseudomonas LPS were reduced in GM(-/-) compared with wild-type (GM(+/+)) mice. Secretion of TNF-alpha by AMs exposed to LPS ex vivo was also reduced in GM(-/-) mice and restored in mice expressing GM-CSF specifically in the lungs (SPC-GM(+/+)/GM(-/-) mice). LPS-dependent NF-kappaB promoter activity, TNF-alpha secretion, and neutrophil chemokine release were reduced in AM cell lines derived from GM(-/-) mice (mAM) compared with GM(+/+) (MH-S). Retroviral expression of PU.1 in mAM cells, which normally lack PU.1, rescued all of these AM defects. To determine whether GM-CSF, via PU.1, regulated expression of TLR-4 pathway components, mRNA and protein levels for key components were evaluated in MH-S cells (GM(+/+), PU.1(Positive)), mAM cells (GM(-/-), PU.1(Negative)), and mAMPU.1+ cells (GM(-/-), PU.1(Positive)). Cluster of differentiation antigen-14, radioprotective 105, IL-1 receptor-associated kinase (IRAK)-M mRNA, and protein were dependent upon GM-CSF and restored by expression of PU.1. In contrast, expression of other TLR-4 pathway components (myeloid differentiation-2, TLR-4, IRAK-1, IRAK-2, Toll/IL-1 receptor domain containing adapter protein/MyD88 adaptor-like, myeloid differentiation primary-response protein 88, IRAK-4, TNF receptor-associated factor-6, NF-kappaB, inhibitor of NF-kappaB kinase) were not GM-CSF or PU.1-dependent. These results show that GM-CSF, via PU.1, enables AM responses to P. aeruginosa LPS by regulating expression of a specific subset of components of the TLR-4 signaling pathway.
Berclaz
Pierre-Yves
PY
Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, OH 45229, USA.
Carey
Brenna
B
Fillipi
Marie-Dominique
MD
Wernke-Dollries
Kara
K
Geraci
Nick
N
Cush
Stephanie
S
Richardson
Terry
T
Kitzmiller
Joe
J
O'connor
Michael
M
Hermoyian
Christina
C
Korfhagen
Thomas
T
Whitsett
Jeffrey A
JA
Trapnell
Bruce C
BC
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D002460:Cell Line; D018925:Chemokines / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000502:physiology*; D053592:Interleukin-1 Receptor-Associated Kinases / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D011550:Pseudomonas aeruginosa; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D015534:Trans-Activators / Q000502:physiology*; D014158:Transcription, Genetic; D000099232:Proto-Oncogene Protein Spi-1
D015703:Antigens, CD; D018925:Chemokines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D053592:Interleukin-1 Receptor-Associated Kinases; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D051197:Toll-Like Receptor 4; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C490500:Ly78 protein, mouse; C493487:Tlr4 protein, mouse; C507772:Irak3 protein, mouse