Cellular homeostasis in response to transition metals associated with shipping emissions

Airborne particulate matter (PM), is associated with almost 9 million premature deaths per year worldwide. PM is regulated by its concentration in the air, but this neglects its composition, which varies depending on source, and is thought to be an important determinant of its health effects. PM from ships contributes to air pollution in port and coastal areas but is rarely studied. Ships often burn heavy oil as fuel, producing PM which I have shown to contain increased concentrations of vanadium, nickel, and cobalt, compared to PM from other sources. These metals are found especially in ultrafine PM (UFPM), less than 100 nanometres in diameter and small enough to reach the air sacs (alveoli) deep in the lungs. Alveoli allow inhaled oxygen to enter the blood, so maintaining healthy alveoli is critical. Alveolar lining cells, called epithelial cells, can heal in response to damage, while damage-causing agents such as bacteria or particles can be enveloped and digested by cells called macrophages. However, little is known about whether these cells function normally in the presence of these different metals. PM exposure is a leading risk factor for ill health over our lives, so understanding how our lungs can stay healthy is of great importance.

I will begin by studying how vanadium, nickel, and cobalt affect the ability of the epithelial cells and macrophages to perform their functions, comparing effects to UFPM collected near a busy shipping area. I will then examine effects on known responses to PM, including activation of defences against metals and release of chemical messages to coordinate responses within and between cells. I will use a technique called transcriptomics, which allows gives information about all the signals within a cell which are altered in response to these metals, meaning previously undocumented responses can be discovered. Thus, these first parts of the project will focus on how exposed cells are affected by the metals. Next I will focus on how the metals are dealt with by the cells, which aim to maintain a state of optimal functioning, a process called homeostasis. I will use a technique called inductively coupled plasma mass spectrometry (ICP-MS) to study the extent to which these metals enter, and are retained within, exposed cells, and whether responses of cells to keep concentrations of these metals under control might also affect concentrations of other vital metals, including iron, copper, and zinc. I will use fluorescent dyes to visualise the changes in concentrations of these metals within cells, and then study the rate at which the cell produces proteins which enable entry, retention, and excretion of the metals, to understand how cells regulate the amount of these metals which they contain.

I will next use cultures of macrophages and epithelial cells together with blood vessel cells, representing the whole alveolus, and then thin slices of whole lung tissue, exposed to these metals, to study transcriptomic changes in individual cells, showing cell-to-cell variation in responses. I will then use state-of-the-art analytical chemistry techniques to study concentrations of metals in individual cells, to see whether some of the ship-associated metals are taken up more by different cells, and whether this affects uptake of other metals critical to the normal functioning of the cells. Finally, I will match the two sets of information together, to understand the relationship between concentrations of a range of metals within cells and the processes activated and deactivated within the same cells.

This project will improve understanding of how these metals in airborne PM might affect our lungs. In turn, this will suggest whether some of these metals should be further studied, to better inform policy to protect the health of the public. This might lead to restrictions in the concentrations of these metals in fuels, or regulations regarding ships' use of engines in port.

Particulate matter (PM) from ships contributes significantly to air pollution in port cities, but little is understood about the effects of its components. Compared to urban PM, shipping PM (especially ultrafine PM <100nm diameter) is enriched in vanadium, nickel, and cobalt, which may drive distinct effects. This project will study the effects of these metals in the alveoli, alone, in combination, and compared to whole PM, testing the hypothesis that shipping emissions dysregulate cellular homeostasis in a manner dependent on the specific metal profile of the PM.

First, effects of these metals on maintenance of healthy alveoli will be studied by exposing type II alveolar epithelial cells (ATII) and macrophages to these metals, and measuring ATII markers of epithelial-mesenchymal transition (EMT-related transcription factors, cell migration), and altered macrophage function (immunophenotype, phagocytic activity). Next, established PM responses (antioxidant expression, inflammatory mediator release, signalling pathway activation) will be measured, and RNA-Seq used to identify transcriptomic changes. Effects on homeostasis of crucial metals (iron, copper, zinc) will be visualised microscopically via metal-sensitive dyes, followed by measurement of intracellular metals by ICP-MS. The function of key metal transporters and regulatory pathways in metal homeostasis will be evaluated by immunocytochemistry and gene knockdown. Finally, more complex models will be used - ATII-ATI-macrophage-endothelial cocultures, using LA-ICP-TOF MS to map metal concentration at single cell resolution, using single-cell RNA-Seq to study heterogeneity in transcriptomic response, followed by precision cut lung slices, pairing LA-ICP-TOF MS and GeoMX transcriptomics to build a correlative metal concentration-transcriptomic lung map.

This work will show how elements enriched in shipping PM may perturb homeostatic mechanisms in the lung, and how this may affect alveolar function.