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Mammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor B

Kaczorowski, DJ;Scott, MJ;Pibris, JP;Afrazi, A;Nakao, A;Edmonds, RD;Kim, S;Kwak, JH;Liu, Y;Fan, J;Billiar, TR;
Product: ULTRA PURE LPS from Escherichia coli O111:B4

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.

PubMed ID: 22526919
232023202017-06-052017-06-0511:03:0211:03:022018-03-142018-03-1411:19:0511:19:05Kaczorowski, DJ;Scott, MJ;Pibris, JP;Afrazi, A;Nakao, A;Edmonds, RD;Kim, S;Kwak, JH;Liu, Y;Fan, J;Billiar, TR;Kaczorowski, DJ;Scott, MJ;Pibris, JP;Afrazi, A;Nakao, A;Edmonds, RD;Kim, S;Kwak, JH;Liu, Y;Fan, J;Billiar, TR;20122012Mammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor BMammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor BThesisThesis851-60851-6018182252691922526919

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.

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.

4.8244.824

Reagents:

... Ultrapure lipopolysaccharide (LPS) (Escherichia coli 0111:B4, TLR4 lig- and) was purchased from List Biological Laboratories, Inc. (Vandell Way, CA, USA). ...

Results - DNA Stimulates Factor B Synthesis and Release by Macrophages:

Early upregulation of factor B occurs after ischemic tissue injury. However, the endogenous molecules that stimulate factor B production in the setting of ischemic tissue injury have not been identified. We have previously shown that microbial ligands, including LPS and polyinosine–polycytidylic acid (poly I:C) stimulate dynamic synthesis and release of factor B by macrophages (26). We next sought to test the hypothesis that one of several known endogenous mediators of inflammation known to activate patternrecognition receptors would stimulate factor B synthesis. Because we previously found that activation of TLR4 signaling by LPS resulted in synthesis of factor B in macrophages, we therefore studied the effect of several endogenous molecules that have been identified as activators of TLR4 signaling (33–35). As previously observed, stimulation of RAW264.7 macrophages with LPS resulted in upregulation of factor B mRNA (9.9-fold, p<0. 001). However, no significant upregulation of factor B was observed after stimulation with fibrinogen, heparan sulfate or HMGB1 at known stimulatory concentrations (Figure 1). ...

Reagents:

... Ultrapure lipopolysaccharide (LPS) (Escherichia coli 0111:B4, TLR4 lig- and) was purchased from List Biological Laboratories, Inc. (Vandell Way, CA, USA). ...

Results - DNA Stimulates Factor B Synthesis and Release by Macrophages:

Early upregulation of factor B occurs after ischemic tissue injury. However, the endogenous molecules that stimulate factor B production in the setting of ischemic tissue injury have not been identified. We have previously shown that microbial ligands, including LPS and polyinosine–polycytidylic acid (poly I:C) stimulate dynamic synthesis and release of factor B by macrophages (26). We next sought to test the hypothesis that one of several known endogenous mediators of inflammation known to activate patternrecognition receptors would stimulate factor B synthesis. Because we previously found that activation of TLR4 signaling by LPS resulted in synthesis of factor B in macrophages, we therefore studied the effect of several endogenous molecules that have been identified as activators of TLR4 signaling (33–35). As previously observed, stimulation of RAW264.7 macrophages with LPS resulted in upregulation of factor B mRNA (9.9-fold, p<0. 001). However, no significant upregulation of factor B was observed after stimulation with fibrinogen, heparan sulfate or HMGB1 at known stimulatory concentrations (Figure 1). ...

https://www.researchgate.net/profile/Amin_Afrazi/publication/224819833_Mammalian_DNA_is_an_endogenous_danger_signal_that_stimulates_local_synthesis_and_release_of_Complement_Factor_B/links/0a85e52fd820760d66000000.pdfhttps://www.researchgate.net/profile/Amin_Afrazi/publication/224819833_Mammalian_DNA_is_an_endogenous_danger_signal_that_stimulates_local_synthesis_and_release_of_Complement_Factor_B/links/0a85e52fd820760d66000000.pdf2012-07-182012-07-1810.2119/molmed.2012.0001110.2119/molmed.2012.00011ULTRA PURE LPS from Escherichia coli O111:B4ULTRA PURE LPS from Escherichia coli O111:B4billiartr@upmc.edubilliartr@upmc.eduActivation;Activator;Analysis;Binding;Biological;Cellular;Chain;Complement;Culture;Dependent;DNA;Escherichia coli;Expression;Factor;Hemorrhagic;Immune System;In vitro;Induce;Interferon;Lipopolysaccharide;List;List Biological;LPS;Mammalian;Molecule;Mouse;mRNA;Protein;Reaction;Reagent;Receptor;Regulatory;Release;Shock;Signal;Sterile;Synthesis;Tissue;Transcription;Ultrapure;ThesisActivation;Activator;Analysis;Binding;Biological;Cellular;Chain;Complement;Culture;Dependent;DNA;Escherichia coli;Expression;Factor;Hemorrhagic;Immune System;In vitro;Induce;Interferon;Lipopolysaccharide;List;List Biological;LPS;Mammalian;Molecule;Mouse;mRNA;Protein;Reaction;Reagent;Receptor;Regulatory;Release;Shock;Signal;Sterile;Synthesis;Tissue;Transcription;Ultrapure;Thesis421421mammalian-dna-is-an-endogenousmammalian-dna-is-an-endogenous