Haemozoin activates NF-kappaB and promotes transcription of pro-inflammatory and matrix-related molecules in human monocytes

Introduction. Enhanced inflammatory response, along with deregulation of balances between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), appear to play a crucial role in clinical course towards complicated malaria, including cerebral malaria. The aim of present study is to clarify the role of malarial pigment (HZ, haemozoin) in the regulation of transcription of pro-inflammatory and matrix-related molecules focusing on the involvement of NF-kappaB pathway. Materials and methods. Human monocytes purified from peripheral blood were fed with HZ for 2 hours and further incubated for additional 0, 2, 4, and 15 hours. In selected experiments, unfed monocytes were treated with rhMIP-1alpha, rhTNFalpha, and rhIL-1beta; alternatively, HZ-fed monocytes were treated with anti-hMIP-1alpha, anti-hTNFalpha and anti-hIL-1beta blocking Abs. Therefore, cell supernatants and lysates were collected and used for following analyses. Cytosolic Ikappa-Balpha protein phosphorylation and degradation were evaluated by western blotting. NF-kappaB complex nuclear translocation was studied by electrophoretic mobility shift assay and confirming western blotting. Short term (0-4 hours after phagocytosis) gene expression analysis of pro-inflammatory molecules was performed through macro-array of a complete panel of cytokines and confirming real-time RT-PCR. Long term (15 hours after phagocytosis) transcription of MMP-9 and TIMP-1 was measured by real-time RT-PCR. Results. After phagocytosis of HZ, the inhibitory cytosolic IkappaBalpha protein was phosphorylated and degraded whereas the NF-kappaB complex migrated to the nuclear fraction. Short term gene expression analysis showed that HZ induced immediately IL-1beta transcription, further followed by additional transcription of eight chemokines (IL-8, ENA-78, GROalpha, GRObeta, GROgamma, MIP-1alpha, MIP-1beta and MCP-1), two cytokines (TNFalpha and IL-1RA), and MMP-9 proteolytic enzyme. Long term studies showed that HZ enhanced mRNA levels of either MMP-9 or TIMP-1, these effects being mimicked by rhMIP-1alpha, rhTNFalpha or rhIL-1beta and abrogated by anti-MIP-1alpha, anti-TNFalpha or anti-IL-1beta blocking Abs. Conclusions. Collectively, the present data suggest that phagocytosis of HZ by human monocytes promotes a cascade of events including NF-kappaB pathway activation, higher transcription of IL-1beta, rapidly endorsed by additional cytokines and chemokines, and further impairment of MMP-9/TIMP-1 balance. These evidence might be useful in order to design an adjunctive therapy with anti-inflammatory drugs and synthetic MMP inhibitors aimed to prevent complicated malaria.