Mitochondrial Reactive Oxygen Species Participate in Signaling Triggered by Heme in Macrophages and upon Hemolysis

Abstract

Hemolysis causes an increase of intravascular heme, oxidative damage, and inflammation in which macrophages play a critical role. In these cells, heme can act as a prototypical damage-associated molecular pattern, inducing TLR4-dependent cytokine production through the MyD88 pathway, independently of TRIF. Heme promotes reactive oxygen species (ROS) generation independently of TLR4. ROS and TNF production contribute to heme-induced necroptosis and inflammasome activation; however, the role of ROS in proinflammatory signaling and cytokine production remains unknown. In this study, we demonstrate that heme activates at least three signaling pathways that contribute to a robust MAPK phosphorylation and cytokine expression in mouse macrophages. Although heme did not induce a detectable Myddosome formation, the TLR4/MyD88 axis was important for phosphorylation of p38 and secretion of cytokines. ROS generation and spleen tyrosine kinase (Syk) activation induced by heme were critical for most proinflammatory signaling pathways, as the antioxidant N-acetyl-L-cysteine and a Syk inhibitor differentially blocked heme-induced ROS, MAPK phosphorylation, and cytokine production in macrophages. Early generated mitochondrial ROS induced by heme was Syk dependent, selectively promoted the phosphorylation of ERK1/2 without affecting JNK or p38, and contributed to CXCL1 and TNF production. Finally, lethality caused by sterile hemolysis in mice required TLR4, TNFR1, and mitochondrial ROS, supporting the rationale to target these pathways to mitigate tissue damage of hemolytic disorders.

Delayed bystander CD8 T cell activation, early immune pathology and persistent dysregulation characterise severe COVID-19

“…While free heme can act as damage-associated molecular pattern and promote ROS formation, the role of heme biosynthesis vs . catabolism in balancing cellular sensitivity to oxidants is complex and context dependent (Prestes et al, 2020). Here, given correlated regulation of heme and OXPHOS pathways in the clinical categories C, D and E, activity of these modules may be interrelated and possibly jointly reflective of dysfunctional mitochondria.…”

The receptor for advanced glycation end products is a sensor for cell‐free heme

“…Further studies are needed to identify whether heme shares the same signaling pathway as AGEs and what the physiological consequences of this competition could be. It has already been shown that heme does not share the same signaling pathway as LPS upon binding to TLR4 [8,[23][24][25].…”

“…Via TLR4, it mediates inflammatory processes such as the synthesis of proinflammatory cytokines, monocyte/macrophage activation, and mobilization of Weibel-Palade bodies from endothelial cells [4][5][6]. The deleterious properties of free heme are not, however, fully explained by its interaction with TLR4, raising questions about the implication of other receptor(s) [7,8].…”

Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

“…Heme acts as a regulator of Toll-like receptor-4 (TLR-4) signaling via MyD88 to regulate pro-inflammatory cytokine (i.e., TNFα) production in macrophages and other target cells [ 107 ]. A combination of antioxidant-sensitive pathways triggered by heme-dependent ROS production via spleen tyrosine kinase ( Syk ) signaling, and TLR4 activation were required for cytokines/chemokines production in macrophages and lethal effects of hemolysis in mice [ 108 ]. Recent studies describe differential effects of pro-inflammatory stimuli (i.e., LPS) on labile heme pools and Hmox1 gene expression in mouse bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs).…”