Abstract
Abstract 1198
Tissue factor (TF), the primary initiator of the coagulation cascade, is normally absent from cells that regularly come in contact with blood such as endothelial cells and monocytes yet could be induced in these cells in a variety of pathological conditions. The aberrant expression of TF by cells of the monocyte/macrophage lineage is thought to be a major contributor to thrombotic disorders and inflammation. Macrophages play a central role in the innate immune response essential for host defense against pathogenic infections. Tuberculosis (TB), a fatal disease caused by Mycobacterium tuberculosis (Mtb), affects nearly one third of the world's population. A number of studies have reported the presence of thrombotic complications, particularly disseminated intravascular coagulation (DIC) in TB patients. However, it is unclear how Mtb infection causes DIC or other thrombotic disorders as mycobacteria are not known to produce endotoxins or exotoxins that otherwise initiate the clotting cascade. In the present study, we have investigated whether Mtb infection induces TF expression in macrophages and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. Exposure of human monocyte-derived macrophages (MDM) to live virulent Mtb H37Rv (at 1 to 5 bacteria/macrophage) or γ-Mtb H37Rv (10 μg/ml) markedly increased TF expression in MDM (5 to 20-fold increase compared to untreated MDM). TF expression in macrophages in response to Mtb is predominantly higher in CD14hi (CD14+) macrophages. Although CD14loCD16hi (CD16+) macrophages also expressed TF in response to Mtb, the level of TF induction in these cells was much lower. γ-Mtb induced TF expression in macrophages much more robustly than E.coli-derived LPS. The γ-Mtb-mediated induction of TF expression in macrophages peaked around 9 h and was sustained throughout 48 h, which markedly varies from the kinetics of LPS-induced TF expression, which peaks between 3–6 h and then reduced down to basal level by 18–24 h. To identify the potential macrophage receptor(s) responsible for the induction of TF by Mtb, MDM were pre-treated with antibodies against CD14 and Toll-like receptors (TLR) before they were exposed to γ-Mtb. Although CD14 antibodies markedly inhibited LPS-induced TF activity, they only suppressed Mtb-induced TF activity minimally. Pretreatment of MDMs with TLR2 or TLR4 antibodies had no significant effect on γ-Mtb-induced TF expression. However, combining the CD14 antibodies together with TLR2 and TLR4 antibodies showed nearly a 50% reduction in γ-Mtb-induced TF activity. These data indicate that a cooperative action of multiple receptors and signaling pathways may be responsible for the robust and sustained induction of TF expression. In order to identify the specific component(s) of γ-Mtb that are responsible for TF induction, we have treated MDMs with various subcellular fractions or purified components derived from Mtb H37Rv. The whole cell lysate of Mtb, cell wall, cell membrane and culture filterate proteins induced TF activity to varying degrees. Of all the purified components tested, the mycobacterial cell wall core component mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) elicited induction of TF protein and activity in the order of mAGP > PIM6 > LM. It is interesting to note that the treatment of MDMs with individual components, mycolic acid, arabinogalactan and peptidoglycan of mAGP complex did not induce TF expression, indicating that the structure of mAGP may be required for recognition by pattern recognition receptors on macrophages. In summary, our data show that Mtb induces TF expression in macrophages, probably through a novel mechanism. Although Mtb-induced TF expression in monocytes/macrohages may lead to systemic thrombotic disorders through the release of TF containing microparticles, it may be the localized and sustained expression of TF on macrophages that internalize the infectious bacteria at the site of infection in lung is responsible for granuloma formation in tuberculosis patients that prevents the spread of the bacteria.
Disclosures:
No relevant conflicts of interest to declare.
Synthesis of a homologous series of galactofuranose-containing mycobacterial arabinogalactan fragments