title: GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS.
abstract: 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.
authors.affiliation: Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, OH 45229, USA.
authors.forename: Pierre-Yves
authors.initials: PY
authors.lastname: Berclaz
authors.forename: Brenna
authors.initials: B
authors.lastname: Carey
authors.forename: Marie-Dominique
authors.initials: MD
authors.lastname: Fillipi
authors.forename: Kara
authors.initials: K
authors.lastname: Wernke-Dollries
authors.forename: Nick
authors.initials: N
authors.lastname: Geraci
authors.forename: Stephanie
authors.initials: S
authors.lastname: Cush
authors.forename: Terry
authors.initials: T
authors.lastname: Richardson
authors.forename: Joe
authors.initials: J
authors.lastname: Kitzmiller
authors.forename: Michael
authors.initials: M
authors.lastname: O'connor
authors.forename: Christina
authors.initials: C
authors.lastname: Hermoyian
authors.forename: Thomas
authors.initials: T
authors.lastname: Korfhagen
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Whitsett
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Trapnell
mesh_terms: 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
chemical_list: 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