Interplay of bacterial endotoxins and transition metals in the inflammatory capacity of airborne particulate matter
28 June 2018
Stadscampus, Promotiezaal "De Grauwzusters" - Lange Snt-Annastraat 7 - 2000 Antwerpen (route: UAntwerpen, Stadscampus
Organization / co-organization:
Department of Bioscience Engineering
PhD defence Serena Moretti - Faculty of Science, Department of Bioscience Engineering
Endotoxins or lipopolysaccharides (LPS) are constituents of the outer membrane of Gram-negative bacteria and are well known for their high pro-inflammatory potency. As a component of particulate matter (PM) with anticipated health implications, this PhD thesis aimed to investigate the relative contribution of endotoxin – associated with other PM components - in inflammatory responses caused by PM in the urban setting of Antwerp.
First, we explored possible improvements for the current methods on collection and detection of ambient endotoxin. With our more microbial-targeted approach using a Coriolis µ sampler, endotoxins were detected at approximately 10-fold higher concentrations in Antwerp (geometric mean 4.49 EU m-3) compared to the urban air of other cities using filter-based samplers (~0.44 EU m-3 in PM10). These endotoxin concentrations were quantified with the recombinant Factor C (rFC) assay, which is a modern and more sustainable version of the gold standard limulus amebocyte lysate (LAL) assay with reduced likelihood of interference from other PM components. Despite these advantages, the induction of inflammatory markers (IL-1β, IL-8, TNFα) by our air pollution samples, as measured in a human macrophage-like U937 cell line was not well correlated to the rFC-determined ambient endotoxin concentrations. Therefore, another bioassay for the detection of endotoxins was applied. This assay was based on the human recognition of endotoxin via the key innate immune receptor Toll-like receptor 4 (TLR4). Samples from the traffic land-use class were found to significantly increase the activation of the TLR4 bioassay, compared to samples from the industrial and green locations, despite all areas having similar rFC-determined endotoxin concentrations. We subsequently explored how transition metals could affect the TLR4 bioassay and PM-related pro-inflammatory responses. Interestingly, especially iron, a well-known and abundant compound of traffic-related air pollution, was found to enhance the TLR4 bioassay, in contrast to nickel and cobalt which was previously reported to directly activate TLR4. Moreover, because PM and air pollution samples are such complex mixtures, we subsequently showed that by dosing purified endotoxins (LPS) with Fe2+, an increase up to 64% in bioactivity of the HEK hTLR4 cell line (EU ml-1) could be found, while Fe2+ without LPS was unable to stimulate a response. Thus, in this PhD thesis we could show that, although endotoxins are typically quantified in ambient air, their toxicity is significantly influenced by the associated particle composition. Especially iron, one of the most abundant transition metals occurring in urban PM, was found to modulate endotoxin activity.