title: Mammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor B.
abstract: Complement factor B plays a critical role in ischemic tissue injury and autoimmunity. Factor B is dynamically synthesized and released by cells outside of the liver, but the molecules that trigger local factor B synthesis and release during endogenous tissue injury have not been identified. We determined that factor B is upregulated early after cold ischemia-reperfusion in mice, using a heterotopic heart transplant model. These data suggested upregulation of factor B by damage-associated molecular patterns (DAMPs), but multiple common DAMPs did not induce factor B in RAW264.7 mouse macrophages. However, exogenous DNA induced factor B mRNA and protein expression in RAW cells in vitro, as well as in peritoneal and alveolar macrophages in vivo. To determine the cellular mechanisms involved in DNA-induced factor B upregulation we then investigated the role of multiple known DNA receptors or binding partners. We stimulated peritoneal macrophages from wild-type (WT), toll-like receptor 9 (TLR9)-deficient, receptor for advanced glycation end products (RAGE)⁻/⁻ and myeloid differentiation factor 88 (MyD88)⁻/⁻ mice, or mouse macrophages deficient in high-mobility group box proteins (HMGBs), DNA-dependent activator of interferon-regulatory factors (DAI) or absent in melanoma 2 (AIM2), with DNA in the presence or absence of lipofection reagent. Reverse transcription-polymerase chain reaction, Western blotting and immunocytochemical analysis were employed for analysis. Synthesis of factor B was independent of TLR9, RAGE, DAI and AIM2, but was dependent on HMGBs, MyD88, p38 and NF-κB. Our data therefore show that mammalian DNA is an endogenous molecule that stimulates factor B synthesis and release from macrophages via HMGBs, MyD88, p38 and NF-κB signaling. This activation of the immune system likely contributes to damage following sterile injury such as hemorrhagic shock and ischemia-reperfusion.
authors.affiliation: Division of Cardiovascular Surgery, University of Pennsylvania, Philadelphia, PA, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kaczorowski
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Scott
authors.forename: John P
authors.initials: JP
authors.lastname: Pibris
authors.forename: Amin
authors.initials: A
authors.lastname: Afrazi
authors.forename: Atsunori
authors.initials: A
authors.lastname: Nakao
authors.forename: Rebecca D
authors.initials: RD
authors.lastname: Edmonds
authors.forename: Sodam
authors.initials: S
authors.lastname: Kim
authors.forename: Joon H
authors.initials: JH
authors.lastname: Kwak
authors.forename: Yujian
authors.initials: Y
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Fan
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
mesh_terms: D000818:Animals; D002460:Cell Line; D011415:Complement Factor B / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D024002:HMGB Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D016601:RNA-Binding Proteins; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: C540422:Aim2 protein, mouse; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D024002:HMGB Proteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D051217:Toll-Like Receptor 9; C402632:Zbp1 protein, mouse; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D011415:Complement Factor B