Hemozoin Increases IFN-γ-Inducible Macrophage Nitric Oxide Generation Through Extracellular Signal-Regulated Kinase- and NF-κB-Dependent Pathways

Abstract

NO overproduction has been suggested to contribute to the immunopathology related to malaria infection. Even though a role for some parasite molecules (e.g., GPI) in NO induction has been proposed, the direct contribution of hemozoin (HZ), another parasite metabolite, remains to be established. Therefore, we were interested to determine whether Plasmodium falciparum (Pf) HZ and synthetic HZ, β-hematin, alone or in combination with IFN-γ, were able to induce macrophage (Mφ) NO synthesis. We observed that neither Pf HZ nor synthetic HZ led to NO generation in B10R murine Mφ; however, they significantly increased IFN-γ-mediated inducible NO synthase (iNOS) mRNA and protein expression, and NO production. Next, by investigating the transductional mechanisms involved in this cellular regulation, we established that HZ induces extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase phosphorylation as well as NF-κB binding to the iNOS promoter, and enhances the IFN-γ-dependent activation of both second messengers. Of interest, cell pretreatment with specific inhibitors against either NF-κB or the ERK1/2 pathway blocked the HZ + IFN-γ-inducible NF-κB activity and significantly reduced the HZ-dependent increase on IFN-γ-mediated iNOS and NO induction. Even though selective inhibition of the Janus kinase 2/STAT1α pathway suppressed NO synthesis in response to HZ + IFN-γ, HZ alone did not activate this signaling pathway and did not have an up-regulating effect on the IFN-γ-induced Janus kinase 2/STAT1α phosphorylation and STAT1α binding to the iNOS promoter. In conclusion, our results suggest that HZ exerts a potent synergistic effect on the IFN-γ-inducible NO generation in Mφ via ERK- and NF-κB-dependent pathways.

Effect of malaria components on blood mononuclear cells involved in immune response

“…Previous studies have proposed that both native P. falciparum hemozoin and synthetic hemozoin can induce activation of ERK 1 and 2 pathways and binding of NF-κB to the murine inducible nitric oxide synthase (iNOS) promoter, rapidly occurring within 1 h of stimulation and returning to almost basal levels after 4 h [21] . The presence of both synthetic hemozoin and interferon (IFN)-γ has been shown to act in synergy to induce NF-κB binding to the murine iNOS gene [21] .…”

“…Previous studies have proposed that both native P. falciparum hemozoin and synthetic hemozoin can induce activation of ERK 1 and 2 pathways and binding of NF-κB to the murine inducible nitric oxide synthase (iNOS) promoter, rapidly occurring within 1 h of stimulation and returning to almost basal levels after 4 h [21] . The presence of both synthetic hemozoin and interferon (IFN)-γ has been shown to act in synergy to induce NF-κB binding to the murine iNOS gene [21] . In addition, ex vivo and in vitro studies have demonstrated that significantly elevated activity of NO synthase (NOS) was found in peripheral blood mononuclear cells (PBMCs) from children with malaria anemia and that it is inversely associated with hemoglobin levels [22,23] , suggesting that hemozoin can induce NOS2 related to NO formation [22] .…”

“…In vitro studies have demonstrated that PRBCs can induce the production of TNF-α, IL-12, and IFN-γ by PBMCs of naive donors within 10 h [33] . Several experimental in vitro studies have proposed that activation of NF-κB in mononuclear cells was triggered by compartments of malaria parasites including hemozoin [20,21] and GPIs [5,6] . This led to up-regulation of NF-κB signaling and nuclear translocation of NF-κB.…”

“…This led to up-regulation of NF-κB signaling and nuclear translocation of NF-κB. Previous investigations demonstrated that hemozoin can mediate activation of NF-κB pathways in macrophages and blood mononuclear cells via rapid phosphorylation of IκBα and subsequent NF-κB nuclear translocation to regulate enhancement of inflammatory cytokines [18,[20][21][22] . In human monocytes fed with trophozoites and hemozoin, persistent activation of NF-κB signaling was connected with activation of matrix metalloproteinase-9 (MMP-9) [18] .…”

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma

“…Recently, several laboratories have reported a correlation between ERK activation and expression of inducible nitric oxide synthase (iNOS) in various nontransformed cells of human, rat, and murine origin (Kristof et al, 2001;Bhat and Fan, 2002;Liu et al, 2002;Jaramillo et al, 2003;Jiang et al, 2004). This cytokine-inducible enzyme is one of the three known nitric oxide synthases, which produce nitric oxide (NO) through the conversion of arginine to citrulline.…”