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

1
Department of Biomedical Sciences, Mercer University School of Medicine, Savannah, GA 31404, USA
2
Department of Biology, Georgia Southern University, Savannah, GA 31419, USA
*
Author to whom correspondence should be addressed.
Biomedicines 2019, 7(2), 41; https://doi.org/10.3390/biomedicines7020041
Received: 16 May 2019 / Accepted: 25 May 2019 / Published: 3 June 2019
(This article belongs to the Special Issue Roles of NF-κB in Cancer and Their Therapeutic Approaches 2019)
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Abstract

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-BlueTM 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 Null2TM 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. View Full-Text
Keywords: NF-kB; hemoglobin; toll-like receptor 4; cancer; reporter cells; immune regulation NF-kB; hemoglobin; toll-like receptor 4; cancer; reporter cells; immune regulation
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Sharma, J.; Boyd, T.; Alvarado, C.; Gunn, E.; Adams, J.; Ness, T.; Dunwoody, R.; Lamb, J.; House, B.; Knapp, J.; Garner, R. Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin. Biomedicines 2019, 7, 41.

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