Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Traceable determination of metal composition of tyres using tandem ICP-MS and benchmarking of emissions inventories.

Non-exhaust emissions are becoming of increasing significance with respect to total particulate matter (PM) concentrations in ambient air. Of particular interest is the metal content of this PM since metallic compounds are well known to have toxic effects on human health and the environment. In this study, 'bottom-up' annual tyre wear emission rates were estimated and compared to top-down' emissions declared by the UK; it was calculated that between 14 and 25 tonnes of Zn entered the atmosphere in PM10 in 2020. The emission rates were estimated using a cost-effective, simple but robust validated method for analysis of the metals in tyres using tandem inductively coupled plasma mass spectrometry (ICP-MS/MS) for the first time, involving minimal offline sample preparation. This method was applied to five different tyre makes and brands, all available for sale in the UK, and the uncertainty of each measurement was determined. Traceability was ensured in all methods and novel validation techniques were applied due to lack of available reference materials. Zn was found to be the largest metal component in all tyres with a mass fraction of approximately 10 mg g-1. The mean mass fractions of metals in the tyres decreased in the order of Zn > Al > Fe > Mg > Ti > Pb > Cu > Ba > Ni. Significant differences in composition were found between the five tyres. The relative expanded uncertainties of the metals measurements ranged from 4 to 21%, with elements of higher mass fraction resulting in lower uncertainties. These findings will contribute to assessing current and future air quality challenges and will help to inform regulation surrounding non-exhaust emissions.

A robust regression analysis method to determine the significance of trends in concentrations of heavy metals in UK ambient air and improve network design and emission inventories.

A novel application of the Theil-Sen robust regression method for determining the temporal trends in the concentration of heavy metals in UK ambient air over the period 2005-2020 is presented and compared to other regression methods. We have demonstrated improvements over non-robust methods of regression, proving the ability to tease out trends that are small with respect to the variability of the concentration measurement. The method is used to identify, in general, large and significant trends in the concentrations of Ni, As, Pb and V over the period 2005-2020, either across the UK as a whole or at groupings of site classifications in the UK. These trends have been compared to trends in emission data determined in the same manner. Although the results for most metals provide confidence that the UK metal network of monitoring sites is successful in appropriately capturing changes in emissions, a key finding of this work is the disagreement between trends in measured concentrations and emissions for Cu, Mn and Ni, for which we suggest improvements in future network design. The results also indicate that UK emission data for V should be reviewed, as we propose that the rate of reduction of V emissions is likely to have been overestimated.

Multi-element analysis of tyre rubber for metal tracers.

The purpose of this study was to identify a characteristic elemental tyre fingerprint that can be utilised in atmospheric source apportionment calculations. Currently zinc is widely used as a single element tracer to quantify tyre wear, however several authors have highlighted issues with this approach. To overcome this, tyre rubber tread was digested and has been analysed for 25 elements by ICP-MS to generate a multielement profile. Additionally, to estimate the percentage of the tyre made up of inert fillers, thermogravimetric analysis was performed on a subset. Comparisons were made between passenger car and heavy goods vehicle tyre composition, and a subset of tyres had both tread and sidewall sampled for further comparison. 19 of the 25 elements were detected in the analysis. The mean mass fraction of zinc detected was 11.17 g/kg, consistent with previous estimates of 1% of the tyre mass. Aluminium, iron, and magnesium were found to be the next most abundant elements. Only one source profile for tyre wear exists in both the US and EU air pollution species profile databases, highlighting the need for more recent data with better coverage of tyre makes and models. This study provides data on new tyres which are currently operating on-road in Europe and is therefore relevant for ongoing atmospheric studies assessing the levels of tyre wear particles in urban areas.

Air-quality networks collect environmental DNA with the potential to measure biodiversity at continental scales.

One of the biggest planetary challenges is the accelerating loss of biodiversity threatening ecosystem functioning on a global scale. The WWF Living Planet Report (https://livingplanet.panda.org/) estimates a 69% decline in populations since 1970. The Convention on Biological Diversity and related international treaties ask countries to monitor shifts in community composition and assess rates of species decline to quantify extant biodiversity relative to global targets1. However, quantifying biodiversity is a challenge, and monitoring continual change is impossible at almost any scale due to a lack of standardized data and indicators2,3. A common problem is that the required infrastructure for such global monitoring does not exist. Here, we challenge this notion by analysing environmental DNA (eDNA) captured along with particulate matter by routine ambient air quality monitoring stations in the UK. In our samples, we identified eDNA from >180 vertebrate, arthropod, plant and fungal taxa representative of local biodiversity. We contend that air monitoring networks are in fact gathering eDNA data reflecting local biodiversity on a continental scale, as a result of their routine function. In some regions, air quality samples are stored for decades, presenting the potential for high resolution biodiversity time series. With minimal modification of current protocols, this material provides the best opportunity to date for detailed monitoring of terrestrial biodiversity using an existing, replicated transnational design and it is already in operation.

Cigarette smoke depletes alveolar macrophages and delays clearance of Legionella pneumophila.

Legionella pneumophila is the main etiological agent of Legionnaires' disease, a severe bacterial pneumonia. L. pneumophila is initially engulfed by alveolar macrophages (AMs) and subvert normal cellular functions to establish a replicative vacuole. Cigarette smokers are particularly susceptible to developing Legionnaires' disease and other pulmonary infections; however, little is known about the cellular mechanisms underlying this susceptibility. To investigate this, we used a mouse model of acute cigarette smoke exposure to examine the immune response to cigarette smoke and subsequent L. pneumophila infection. Contrary to previous reports, we show that cigarette smoke exposure alone causes a significant depletion of AMs using enzymatic digestion to extract cells, or via imaging intact lung lobes by light-sheet microscopy. Furthermore, treatment of mice deficient in specific types of cell death with smoke suggests that NLRP3-driven pyroptosis is a contributor to smoke-induced death of AMs. After infection, smoke-exposed mice displayed increased pulmonary L. pneumophila loads and developed more severe disease compared with air-exposed controls. We tested if depletion of AMs was related to this phenotype by directly depleting them with clodronate liposomes and found that this also resulted in increased L. pneumophila loads. In summary, our results showed that cigarette smoke depleted AMs from the lung and that this likely contributed to more severe Legionnaires' disease. Furthermore, the role of AMs in L. pneumophila infection is more nuanced than simply providing a replicative niche, and our studies suggest they play a major role in bacterial clearance.

Role of Secondary Organic Matter on Soot Particle Toxicity in Reconstituted Human Bronchial Epithelia Exposed at the Air-Liquid Interface.

Secondary organic matter (SOM) formed from gaseous precursors constitutes a major mass fraction of fine particulate matter. However, there is only limited evidence on its toxicological impact. In this study, air-liquid interface cultures of human bronchial epithelia were exposed to different series of fresh and aged soot particles generated by a miniCAST burner combined with a micro smog chamber (MSC). Soot cores with geometric mean mobility diameters of 30 and 90 nm were coated with increasing amounts of SOM, generated from the photo-oxidation of mesitylene and ozonolysis of α-pinene. At 24 h after exposure, the release of lactate dehydrogenase (LDH), indicating cell membrane damage, was measured and proteome analysis, i.e. the release of 102 cytokines and chemokines to assess the inflammatory response, was performed. The data indicate that the presence of the SOM coating and its bioavailability play an important role in cytotoxicity. In particular, LDH release increased with increasing SOM mass/total particle mass ratio, but only when SOM had condensed on the outer surface of the soot cores. Proteome analysis provided further evidence for substantial interference of coated particles with essential properties of the respiratory epithelium as a barrier as well as affecting cell remodeling and inflammatory activity.

Falling nickel concentrations in ambient air in South Wales - 50 years of progress.

Measurement of the composition of ambient air has become increasingly widespread over the last 50 years as the detrimental health effects of some air pollutants have become clearer and requirements for these measurements has been embedded in national and international legislation. The aim of this has been not only to assess exposure of the general population to air pollutants but also to assess the effectiveness of abatement strategies to reduce emissions of these pollutants at source. With a rich industrial heritage, the Swansea Valley (South Wales, UK) has long been associated with the refining and production of metal products, especially nickel. Despite a decline in output during the latter part of the twentieth century there is still sufficient activity to prompt a requirement for targeted air monitoring in the area. This is most important for nickel where there is a local history of measured concentrations exceeding legislative target values. This work demonstrates the effectiveness of nickel emissions abatement strategies over the last 50 years by tracking the falling air concentration of nickel over this period. It also demonstrates how the monitoring network in the Swansea Valley has expanded over this time and become significantly more sensitive to nickel emissions. The data presented represents a significant public health achievement - it is likely that the exposure to nickel in air of the population in the Swansea Valley has decreased more than 100-fold over the last 50 years - and reflects the progress in regulation, industrial efficiency, emissions abatement technology and air quality monitoring science achieved during this period.

Emerging Paradigms in Type 2 Immunity.

A principal purpose of type 2 immunity was thought to be defense against large parasites, but it also functions in the restoration of homeostasis, such as toxin clearance following snake bites. In other cases, like allergy, the type 2 T helper (Th2) cytokines and cells present in the environment are detrimental and cause diseases. In recent years, the recognition of cell heterogeneity within Th2-associated cell populations has revealed specific functions of cells with a particular phenotype or gene signature. In addition, here we discuss the recent data regarding heterogeneity of type 2 immunity-related cells, as well as their newly identified role in a variety of processes ranging from involvement in respiratory viral infections [especially in the context of the recent COVID-19 (coronavirus disease 2019) pandemic] to control of cancer development or of metabolic homeostasis.

IFNγ receptor down-regulation facilitates Legionella survival in alveolar macrophages.

Legionella pneumophila is an opportunistic human pathogen and causative agent of the acute pneumonia known as Legionnaire's disease. Upon inhalation, the bacteria replicate in alveolar macrophages (AM), within an intracellular vacuole termed the Legionella-containing vacuole. We recently found that, in vivo, IFNγ was required for optimal clearance of intracellular L. pneumophila by monocyte-derived cells (MC), but the cytokine did not appear to influence clearance by AM. Here, we report that during L. pneumophila lung infection, expression of the IFNγ receptor subunit 1 (IFNGR1) is down-regulated in AM and neutrophils, but not MC, offering a possible explanation for why AM are unable to effectively restrict L. pneumophila replication in vivo. To test this, we used mice that constitutively express IFNGR1 in AM and found that prevention of IFNGR1 down-regulation enhanced the ability of AM to restrict L. pneumophila intracellular replication. IFNGR1 down-regulation was independent of the type IV Dot/Icm secretion system of L. pneumophila indicating that bacterial effector proteins were not involved. In contrast to previous work, we found that signaling via type I IFN receptors was not required for IFNGR1 down-regulation in macrophages but rather that MyD88- or Trif- mediated NF-κB activation was required. This work has uncovered an alternative signaling pathway responsible for IFNGR1 down-regulation in macrophages during bacterial infection.

Cooperative adaptation to therapy (CAT) confers resistance in heterogeneous non-small cell lung cancer.

Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model's parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy.

Plasmacytoid Dendritic Cells Provide Protection Against Bacterial-Induced Colitis.

We have examined the influence of depleting plasmacytoid dendritic cells (pDC) in mice on the immune response to the gut pathogen Citrobacter rodentium, an organism that is a model for human attaching effacing pathogens such as enterohaemorraghic E. coli. A significantly higher number of C. rodentium were found in mice depleted of pDC from 7 days after infection and pDC depleted mice showed increased gut pathology and higher levels of mRNA encoding inflammatory cytokines in the colon upon infection. pDC-depletion led to a compromising of the gut mucosal barrier that may have contributed to increased numbers of C. rodentium in systemic organs. pDC-depleted mice infected with C. rodentium suffered substantial weight loss necessitating euthanasia. A number of observations suggested that this was not simply the result of dysregulation of immunity in the colon as pDC-depleted mice infected intravenously with C. rodentium also exhibited exacerbated weight loss, arguing that pDC influence systemic immune responses. Overall, these data indicate that pDC contribute at multiple levels to immunity to C. rodentium including control of bacterial numbers in the colon, maintenance of colon barrier function and regulation of immune responses to disseminated bacteria.

Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

Identification and characterization of an hnRNP E1 translational silencing motif.

Non-canonical transforming growth factor ß (TGFß) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3' untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained. This study characterizes the binding kinetics of the consensus-binding motif and hnRNP E1, its various K-homology (KH) domains and p-hnRNP E1. Levels of p-hnRNP E1 are highly upregulated in metastatic cancer cells and low in normal epithelial tissue. We show a correlation between this PTM and levels of Akt2 and its activated form, phosphorylated serine-474 (p-Akt2). Using cellular progression models of metastasis, we observed a signature high level of Akt2, p-Akt2 and p-hnRNP E1 protein expression, coupled to a significantly reduced level of total hnRNP E1 in metastatic cells. Genes that are translationally silenced by hnRNP E1 and expressed by its dissociation are highly implicated in the progression of EMT and metastasis. This study provides insight into a non-canonical TGFß signaling cascade that is responsible for inducing EMT by aberrant expression of hnRNP E1 silenced targets. The relevance of this system in metastatic progression is clearly shown in cellular models by the high abundance of p-hnRNP E1 and low levels of hnRNP E1. New insights provided by the resolution of this molecular mechanism provide targets for therapeutic intervention and give further insight into the role of the TGFß microenvironment.

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Suitability of different containers for the sampling and storage of biogas and biomethane for the determination of the trace-level impurities--A review.

The traceable and accurate measurement of biogas impurities is essential in order to robustly assess compliance with the specifications for biomethane being developed by CEN/TC408. An essential part of any procedure aiming to determinate the content of impurities is the sampling and the transfer of the sample to the laboratory. Key issues are the suitability of the sample container and minimising the losses of impurities during the sampling and analysis process. In this paper, we review the state-of-the-art in biogas sampling with the focus on trace impurities. Most of the vessel suitability studies reviewed focused on raw biogas. Many parameters need to be studied when assessing the suitability of vessels for sampling and storage, among them, permeation through the walls, leaks through the valves or physical leaks, sorption losses and adsorption effects to the vessel walls, chemical reactions and the expected initial concentration level. The majority of these studies looked at siloxanes, for which sampling bags, canisters, impingers and sorbents have been reported to be fit-for-purpose in most cases, albeit with some limitations. We conclude that the optimum method requires a combination of different vessels to cover the wide range of impurities commonly found in biogas, which have a wide range of boiling points, polarities, water solubilities, and reactivities. The effects from all the parts of the sampling line must be considered and precautions must be undertaken to minimize these effects. More practical suitability tests, preferably using traceable reference gas mixtures, are needed to understand the influence of the containers and the sampling line on sample properties and to reduce the uncertainty of the measurement.

Computational Identification of Post Translational Modification Regulated RNA Binding Protein Motifs.

RNA and its associated RNA binding proteins (RBPs) mitigate a diverse array of cellular functions and phenotypes. The interactions between RNA and RBPs are implicated in many roles of biochemical processing by the cell such as localization, protein translation, and RNA stability. Recent discoveries of novel mechanisms that are of significant evolutionary advantage between RBPs and RNA include the interaction of the RBP with the 3' and 5' untranslated region (UTR) of target mRNA. These mechanisms are shown to function through interaction of a trans-factor (RBP) and a cis-regulatory element (3' or 5' UTR) by the binding of a RBP to a regulatory-consensus nucleic acid motif region that is conserved throughout evolution. Through signal transduction, regulatory RBPs are able to temporarily dissociate from their target sites on mRNAs and induce translation, typically through a post-translational modification (PTM). These small, regulatory motifs located in the UTR of mRNAs are subject to a loss-of-function due to single polymorphisms or other mutations that disrupt the motif and inhibit the ability to associate into the complex with RBPs. The identification of a consensus motif for a given RBP is difficult, time consuming, and requires a significant degree of experimentation to identify each motif-containing gene on a genomic scale. We have developed a computational algorithm to analyze high-throughput genomic arrays that contain differential binding induced by a PTM for a RBP of interest-RBP-PTM Target Scan (RPTS). We demonstrate the ability of this application to accurately predict a PTM-specific binding motif to an RBP that has no antibody capable of distinguishing the PTM of interest, negating the use of in-vitro exonuclease digestion techniques.

An investigation of the matrix sensitivity of refinery gas analysis using gas chromatography with flame ionisation detection.

The response of a flame ionisation detector (FID) on a gas chromatograph to methane, ethane, propane, i-butane and n-butane in a series of multi-component refinery gas standards was investigated to assess the matrix sensitivity of the instrument. High-accuracy synthetic gas standards, traceable to the International System of Units, were used to minimise uncertainties. The instrument response exhibited a small dependence on the component amount fraction: this behaviour, consistent with that of another FID, was thoroughly characterised over a wide range of component amount fractions and was shown to introduce a negligible bias in the analysis of refinery gas samples, provided a suitable reference standard is employed. No significant effects of the molar volume, density and viscosity of the gas mixtures on the instrument response were observed, indicating that the FID is suitable for the analysis of refinery gas mixtures over a wide range of component amount fractions provided that appropriate drift-correction procedures are employed.