nHZ induces immunostimulatory effects in human monocytes. (A) ROS release (oxidative burst) from monocytes after the addition of nHZ. Suspended nonprimed monocytes were supplemented with nHZ at 50 RBC equivalents in terms of heme-content/monocyte or RBCs treated with anti-D IgG at 50 RBCs/monocyte immediately after isolation of PBMCs from healthy donors at time 0 or kept as unfed control (CTRL) and incubated at 37°C. Aliquots of 1.5 × 10⁵ monocytes were taken at indicated times, and ROS was quantified by luminol-enhanced luminescence. Results from 12 independent experiments are shown. (B) TNF and (C) MCP-1 release from adherent monocytes after the addition of nHZ. Monocytes obtained from healthy donors were enriched by adherence of PBMCs. Approximately 10⁶ monocytes per well were cultured in 1 mL of serum-free MSFM overnight in the presence of 200 U/mL IFNγ and then supplemented with either nHZ (100nmol/10⁶ monocytes in terms of heme content) or nonparasitized RBCs opsonized with anti-D IgG (100 RBCs/monocyte) or kept as unfed control (CTRL). After 3 hours, HZ- and cell-free supernatants were collected and analyzed for TNF and MCP-1 by ELISA. Seven and 5 independent experiments were conducted for TNF and MCP-1 analysis, respectively. All results are expressed as medians with 95% CI. P values were obtained comparing each nHZ-fed sample with its relative nonfed (CTRL) and RBC-fed control. *P < .05, and #P < .01.