Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.
Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates Toll-Like Receptor (TLR) signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and lipopolysaccharide (LPS)-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, and wild-type and mutant proteins, we show that Flii is secreted by means of a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine-rich repeat domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii-depleted conditioned medium leads to enhanced macrophage activation and increased TNF secretion compared with cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.
CHBRI Wound Healing Laboratory, Kids Research Institute, Children's Hospital at Westmead, Sydney, NSW 2145,  Australia.
Nazi
N
Lei
Linda
L
Franken
Nadira
N
Ruzehaji
Carolin
C
Offenhäuser
Allison J
AJ
Cowin
Rachael Z
RZ
Murray
D000818:Animals; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008262:Macrophage Activation; D008561:Membrane Fusion; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011485:Protein Binding; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012867:Skin / Q000378:metabolism; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D002352:Carrier Proteins; D016207:Cytokines; D003598:Cytoskeletal Proteins; C098179:FLII protein, human; C000609263:FlII protein, mouse; D008070:Lipopolysaccharides; D052118:Lysosomal-Associated Membrane Protein 1; D008840:Microfilament Proteins; D050765:Qa-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C541731:Stx11 protein, mouse; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C000716465:rab7 GTP-binding proteins, mouse; D020691:rab GTP-Binding Proteins