FIG. 4.

Heme-induced ROS and ER stress in microglial cells requires TLR4. (A) TLR4 transcripts in spinal microglial cells from HbSS-BERK mice (6.31 ± 0.2 months old, n = 6) incubated with or without hemin (40 μM) for 1 h, normalized to GAPDH and relative to transcripts in unstimulated cells (n = 8/treatment). (B) Hemin-induced ROS requires TLR4. Hemin-induced ROS was attenuated in isolated microglial cells from adult HbSS-BERK and HbAA-BERK mice by pretreatment with LPS-RS (100 ng/mL; L), TAK-242 (400 nM; T), or salubrinal (5 μM; S) overnight before hemin (40 μM; H) for 1 h (n = 6 independent experiments). (C, D) Hemin-induced ER stress requires TLR4. Hemin-induced expression of sXBP1 and CHOP (markers of increased ER stress) was attenuated in isolated microglial cells from adult HbAA-BERK (C) and HbSS-BERK (D) mice by TLR4 inhibition. Microglial cells were incubated with Veh, hemin (40 μM; H), hemin with 100 ng/mL LPS-RS (H+L), or LPS-RS (L) for 60 min. At the end of the incubation period, sXBP1 and CHOP protein expression was evaluated by immunoblotting. Density of individual protein bands is normalized to GAPDH and is presented relative to vehicle. Relative density of vehicle was given an arbitrary value of 100. Data are mean ± SEM (n = 3 independent experiments). H: hemin; L: LPS-RS (TLR4 inhibitor); S: salubrinal; T: TAK-242 (TLR4 inhibitor). ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LPS-RS, lipopolysaccharide from Rhodobacter sphaeroides; ROS, reactive oxygen species; sXBP1, spliced XBP1. Color images are available online.