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Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin

Hemoglobin (Hb) released during red blood cell lysis can initiate TLR4-dependent signaling and trigger NF-κB activation in surrounding cells. Observations of chronic bleeding in various cancers leads us to hypothesize that Hb and Hb degradation products released from lysed RBC near cancer nests migh...

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Autores principales: Sharma, Jill, Boyd, Taylor, Alvarado, Claudia, Gunn, Edwin, Adams, Jaimie, Ness, Traci, Dunwoody, Robert, Lamb, John, House, Brittany, Knapp, James, Garner, Ronald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630411/
https://www.ncbi.nlm.nih.gov/pubmed/31163699
http://dx.doi.org/10.3390/biomedicines7020041
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author Sharma, Jill
Boyd, Taylor
Alvarado, Claudia
Gunn, Edwin
Adams, Jaimie
Ness, Traci
Dunwoody, Robert
Lamb, John
House, Brittany
Knapp, James
Garner, Ronald
author_facet Sharma, Jill
Boyd, Taylor
Alvarado, Claudia
Gunn, Edwin
Adams, Jaimie
Ness, Traci
Dunwoody, Robert
Lamb, John
House, Brittany
Knapp, James
Garner, Ronald
author_sort Sharma, Jill
collection PubMed
description Hemoglobin (Hb) released during red blood cell lysis can initiate TLR4-dependent signaling and trigger NF-κB activation in surrounding cells. Observations of chronic bleeding in various cancers leads us to hypothesize that Hb and Hb degradation products released from lysed RBC near cancer nests might modulate local TLR4-positive cells. We addressed the hypothesis in vitro by measuring Hb- and biliverdin (Bv)-induced NF-κB signaling in an engineered human TLR4 reporter cell model (HEK-Blue(TM) hTLR4). Therein, TLR4 stimulation was assessed by measuring NF-κB-dependent secreted alkaline phosphatase (SEAP). hTLR4 reporter cells incubated with 8 ηM lipopolysaccharide (LPS) or 20-40 μM fungal mannoprotein (FM) produced significant amounts of SEAP. hTLR4 reporter cells also produced SEAP in response to human, but not porcine or bovine, Hb. HEK-Blue Null2(TM) reporter cells lacking TLR4 did not respond to LPS, FM, or Hb. Bv was non-stimulatory in reporter cells. When Bv was added to Hb-stimulated reporter cells, SEAP production was reduced by 95%, but when Bv was applied during LPS and FM stimulation, SEAP production was reduced by 33% and 27%, respectively. In conclusion, Hb initiated NF-κB signaling that was dependent upon TLR4 expression and that Bv can act as a TLR4 antagonist. Moreover, this study suggests that hemorrhage and extravascular hemolysis could provide competitive Hb and Bv signaling to nearby cells expressing TLR4, and that this process could modulate NF-κB signaling in TLR4-positive cancer cells and cancer-infiltrating leukocytes.
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spelling pubmed-66304112019-08-19 Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin Sharma, Jill Boyd, Taylor Alvarado, Claudia Gunn, Edwin Adams, Jaimie Ness, Traci Dunwoody, Robert Lamb, John House, Brittany Knapp, James Garner, Ronald Biomedicines Article Hemoglobin (Hb) released during red blood cell lysis can initiate TLR4-dependent signaling and trigger NF-κB activation in surrounding cells. Observations of chronic bleeding in various cancers leads us to hypothesize that Hb and Hb degradation products released from lysed RBC near cancer nests might modulate local TLR4-positive cells. We addressed the hypothesis in vitro by measuring Hb- and biliverdin (Bv)-induced NF-κB signaling in an engineered human TLR4 reporter cell model (HEK-Blue(TM) hTLR4). Therein, TLR4 stimulation was assessed by measuring NF-κB-dependent secreted alkaline phosphatase (SEAP). hTLR4 reporter cells incubated with 8 ηM lipopolysaccharide (LPS) or 20-40 μM fungal mannoprotein (FM) produced significant amounts of SEAP. hTLR4 reporter cells also produced SEAP in response to human, but not porcine or bovine, Hb. HEK-Blue Null2(TM) reporter cells lacking TLR4 did not respond to LPS, FM, or Hb. Bv was non-stimulatory in reporter cells. When Bv was added to Hb-stimulated reporter cells, SEAP production was reduced by 95%, but when Bv was applied during LPS and FM stimulation, SEAP production was reduced by 33% and 27%, respectively. In conclusion, Hb initiated NF-κB signaling that was dependent upon TLR4 expression and that Bv can act as a TLR4 antagonist. Moreover, this study suggests that hemorrhage and extravascular hemolysis could provide competitive Hb and Bv signaling to nearby cells expressing TLR4, and that this process could modulate NF-κB signaling in TLR4-positive cancer cells and cancer-infiltrating leukocytes. MDPI 2019-06-03 /pmc/articles/PMC6630411/ /pubmed/31163699 http://dx.doi.org/10.3390/biomedicines7020041 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sharma, Jill
Boyd, Taylor
Alvarado, Claudia
Gunn, Edwin
Adams, Jaimie
Ness, Traci
Dunwoody, Robert
Lamb, John
House, Brittany
Knapp, James
Garner, Ronald
Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title_full Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title_fullStr Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title_full_unstemmed Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title_short Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin
title_sort reporter cell assessment of tlr4-induced nf-κb responses to cell-free hemoglobin and the influence of biliverdin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630411/
https://www.ncbi.nlm.nih.gov/pubmed/31163699
http://dx.doi.org/10.3390/biomedicines7020041
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