Macrophage inflammatory protein-1alpha mediates matrix metalloproteinase-9 enhancement in human adherent monocytes fed with malarial pigment.

Abstract Objective To investigate the role of macrophage inflammatory protein-1alpha (MIP-1alpha) in the detrimental enhancement of matrix metalloproteinase-9 (MMP-9) expression, release and activity induced by phagocytosis of malarial pigment (haemozoin, HZ) in human monocytes. Methods Human adherent monocytes were unfed/fed with native HZ for 2 h. After 24 hours, MIP-1alpha production was evaluated by ELISA in cell supernatants. Alternatively, HZ-unfed/fed monocytes were treated in presence/absence of anti-human MIP-1alpha blocking antibodies or recombinant human MIP-1alpha for 15 h (RNA studies) or 24 h (protein studies); therefore, MMP-9 mRNA expression was evaluated in cell lysates by Real Time RT-PCR, whereas proMMP-9 and active MMP-9 protein release were measured in cell supernatants by Western blotting and gelatin zymography. Results Phagocytosis of HZ by human monocytes increased production of MIP-1 alpha, mRNA expression of MMP-9 and protein release of proMMP-9 and active MMP-9. All the HZ-enhancing effects on MMP-9 were abrogated by anti-human MIP-1alpha blocking antibodies and mimicked by recombinant human MIP-1alpha. Conclusions The present work suggests a role for MIP-1alpha in the HZ-dependent enhancement of MMP-9 expression, release and activity observed in human monocytes, highlighting new detrimental effects of HZ-triggered proinflammatory response by phagocytic cells in falciparum malaria

Haemozoin Induces Early Cytokine-Mediated Lysozyme Release from Human Monocytes through p38 MAPK- and NF-kappaB- Dependent Mechanisms

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1 beta/MIP-1 alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaB alpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1 beta and MIP-1 alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria

Malarial pigment enhances heat shock protein-27 in THP-1 cells: New perspectives for in vitro studies on monocyte apoptosis prevention

Abstract Objective To investigate the effect of malarial pigment (hemozoin, HZ) on expression of heat shock proteins (HSPs) and cell viability in human monocytes by using a stable cell line (THP-1 cells). Methods THP-1 cells were fed with native HZ or treated with pro-apoptotic molecule gliotoxin for 9 h. Thereafter, the protein expression of HSP-27 and HSP-70 was evaluated by western blotting. Alternatively, HZ-fed cells were cultured up to 72 h and cell viability parameters (survival, apoptosis and necrosis rates) were measured by flow cytometric analysis. Results HZ increased basal protein levels of HSP-27 without altering those of HSP-70 in THP-1 cells, and promoted long-term cell survival without inducing apoptosis. As expected, gliotoxin inhibited HSP-27 protein expression and promoted long-term cell apoptosis. Conclusions Present data show that HZ prevents cell apoptosis and enhances the expression of anti-apoptotic HSP-27 in THP-1 cells, confirming the previous evidences obtained from HZ-fed immunopurified monocytes. Since the use of a stable cell line is pivotal to perform HSP-27 silencing experiments, monocytic THP-1 cells could be a good candidate line for such an approach, which is heavily required to clarify the role of HSP-27 in survival of impaired HZ-fed monocytes during falciparum malaria

Oxygen-loaded nanodroplets effectively abrogate hypoxia dysregulating effects on secretion of MMP-9 and TIMP-1 by human monocytes

Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation