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 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.

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40-50 µg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP's ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.