Fig 1. HEK-blue TLR9 reporter cell system.

(A) Time response of TLR9 activity in HEK cells exposed to HbFe²⁺ (25 uM heme) HbFe²⁺ + GOX, or HbFe²⁺ + SOD & CAT (150 units). (B) Dose response to TLR9 activity in HEK cells exposed for 24 h to HbFe²⁺ (1–50 uM heme). (C) Dose response to TLR9 activity in HEK cells exposed to HbFe²⁺ + GOX. (D) Spectral analysis of catalase free HbFe²⁺ at time zero. (E,F,G) The percent of ferrous (HbFe²⁺) and ferric (HbFe³⁺) in the culture supernatant between 1 and 24 h for HEK reporter cells exposed to HbFe²⁺, HbFe²⁺ + GOX, or Hb Fe²⁺ + SOD & CAT. (H) HEK TLR9 reporter cells treated for 24 h with CpG (20 ug/ml) in the presence or absence of chloroquine. (I) HEK TLR9 reporter cells treated with ferrous Hb (25 uM heme) with and without GOX in the presence or absence of chloroquine. * p<0.01 vs. CTRL or chloroquine treated cells. **p = 0.008 vs CTRL. Hb- hemoglobin, GOX- glucose oxidase, SOD- superoxide dismutase, CAT- catalase. Experiments were completed with a minimum of 8 individual cell preparations per group / per day and run on two separate days (n = 16 per group).