To Örebro University

Due to their exceptional properties and cost effectiveness, polyamides or nylons have emerged as widely used materials, revolutionizing diverse industries, including industrial 3D printing or additive manufacturing (AM). Powder-based AM technologies employ tonnes of polyamide microplastics to produce complex components every year. However, the lack of comprehensive toxicity assessment of particulate polyamides and polyamide-associated chemicals, especially in the light of the global microplastics crisis, calls for urgent action. This study investigated the physicochemical properties of polyamide-12 microplastics used in AM, and assessed a number of toxicity endpoints focusing on inflammation, immunometabolism, genotoxicity, aryl hydrocarbon receptor (AhR) activation, endocrine disruption, and cell morphology. Specifically, microplastics examination by means of field emission scanning electron microscopy revealed that work flow reuse of material created a fraction of smaller particles with an average size of 1-5 µm, a size range readily available for uptake by human cells. Moreover, chemical analysis by means of gas chromatography high-resolution mass spectrometry detected several polyamide-associated chemicals including starting material, plasticizer, thermal stabilizer/antioxidant, and migrating slip additive. Even if polyamide particles and chemicals did not induce an acute inflammatory response, repeated and prolonged exposure of human primary macrophages disclosed a steady increase in the levels of proinflammatory chemokine Interleukin-8 (IL-8/CXCL-8). Moreover, targeted metabolomics disclosed that polyamide particles modulated the kynurenine pathway and some of its key metabolites. The p53-responsive luciferase reporter gene assay showed that particles per se were able to activate p53, being indicative of a genotoxic stress. Polyamide-associated chemicals triggered moderate activation of AhR and elicited anti-androgenic activity. Finally, a high-throughput and non-targeted morphological profiling by Cell Painting assay outlined major sites of bioactivity of polyamide-associated chemicals and indicated putative mechanisms of toxicity in the cells. These findings reveal that the increasing use of polyamide microplastics may pose a potential health risk for the exposed individuals, and it merits more attention.

Purpose: Occupational particle exposure constitutes a known health hazard in many occupations, while the risks are still largely unknown in more recent industries, like additive manufacturing. The purpose of this study was to investigate how particle exposure in different metal industries affects blood biomarkers known to indicate biological effects on lungs, cardiovascular system, liver, kidneys, and inflammation. The aim was also to understand if particle exposures from different work environments induce similar or distinct biological responses.

Methods: Five cohorts with particle exposure measurement and biomarker data were included in the study: two iron foundry cohorts (40 and 85 participants), one welding cohort (136 participants), one hard metal industry cohort (72 participants), and one additive manufactur-ing cohort (87 participants). Individual dust exposure levels were calculated based on respirable and on inhalable dust exposure measurements, adjusted for respirator use. Biomarker levels were compared to i) control groups vs. exposed individuals within each cohort, or ii) correlated to exposure across all cohorts. The five cohorts were combined into one comprehensive analysis to find common biomarkers of exposure. Biomarker levels were normalized across cohorts using the z-transform based on the control groups, and the normalized biomarker data were correlated to particle exposure levels. Kendall τ correlation was used without covariate correction, and Pearsson correlation as well as ANOVA analysis was used with covariates (age, BMI, sex, smoking, cohort).

The biomarkers were conceptually categorized into six groups, including biomarkers of lung injury (mucin 1, surfactant protein D, CC16, MMP7), cardiovascular impact (ApoA1, ApoB, ApoB/ApoA1 ratio, sST2, vWF, PON1 activity), liver toxicity (ASAT, ALAT, ALP), kidney toxicity (urinary a1 microglobulin), NLRP3 inflammasome activation (IL-1b, IL-18, IL-1Ra), and general inflammation (CRP, SAA, MIP4, sRAGE).

Results: The highest mean respirator adjusted dust exposure was found in the foundry cohorts, followed by additive manufacturing, welding, and finally hard metal industry. When comparing biomarkers of controls and exposed for the cohorts, cardiovascular markers were the once that were most consistently significantly different using Wilcoxon-test, with three out of five cohorts demonstrating differences in ApoB/ApoA1-ratio and sST2-levels. When comparing dust exposure to biomarkers across all cohorts, ALAT was significantly correlated to exposure using Kendall τ (p 0.03) as well as in ANOVA analysis, including correction for covariates (p 0.03). These results indicate that metal dust exposure may put a stress on the cardiovascular system and liver, regardless of metal industry exposure, and that exposure to secondary organs, during clearing of the particles from the lungs, is an important health aspect to consider in regard to exposure of inhaled metal particles.

The NLRP3 inflammasome is a key regulator of inflammation that responds to a broad range of stimuli. The exact mechanism of activation has not been determined, but there is a consensus on cellular potassium efflux as a major common denominator. Once NLRP3 is activated, it forms high-order complexes together with NEK7 that trigger aggregation of ASC into specks. Typically, there is only one speck per cell, consistent with the proposal that specks form - or end up at - the centrosome. ASC polymerisation in turn triggers caspase-1 activation, leading to maturation and release of IL-1β and pyroptosis, i.e., highly inflammatory cell death. Several gain-of-function mutations in the NLRP3 inflammasome have been suggested to induce spontaneous activation of NLRP3 and hence contribute to development and disease severity in numerous autoinflammatory and autoimmune diseases. Consequently, the NLRP3 inflammasome is of significant clinical interest, and recent attention has drastically improved our insight in the range of involved triggers and mechanisms of signal transduction. However, despite recent progress in knowledge, a clear and comprehensive overview of how these mechanisms interplay to shape the system level function is missing from the literature. Here, we provide such an overview as a resource to researchers working in or entering the field, as well as a computational model that allows for evaluating and explaining the function of the NLRP3 inflammasome system from the current molecular knowledge. We present a detailed reconstruction of the molecular network surrounding the NLRP3 inflammasome, which account for each specific reaction and the known regulatory constraints on each event as well as the mechanisms of drug action and impact of genetics when known. Furthermore, an executable model from this network reconstruction is generated with the aim to be used to explain NLRP3 activation from priming and activation to the maturation and release of IL-1β and IL-18. Finally, we test this detailed mechanistic model against data on the effect of different modes of inhibition of NLRP3 assembly. While the exact mechanisms of NLRP3 activation remains elusive, the literature indicates that the different stimuli converge on a single activation mechanism that is additionally controlled by distinct (positive or negative) priming and licensing events through covalent modifications of the NLRP3 molecule. Taken together, we present a compilation of the literature knowledge on the molecular mechanisms on NLRP3 activation, a detailed mechanistic model of NLRP3 activation, and explore the convergence of diverse NLRP3 activation stimuli into a single input mechanism.

Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

Micro- and nanoplastics (MNPs) are emerging pollutants with scarcely investigated effects on human innate immunity. If they follow a similar course of action as other, more thoroughly investigated particulates, MNPs may penetrate epithelial barriers, potentially triggering a cascade of signaling events leading to cell damage and inflammation. Inflammasomes are intracellular multiprotein complexes and stimulus-induced sensors critical for mounting inflammatory responses upon recognition of pathogen- or damage-associated molecular patterns. Among these, the NLRP3 inflammasome is the most studied in terms of activation via particulates. However, studies delineating the ability of MNPs to affect NLRP3 inflammasome activation are still rare. In this review, we address the issue of MNPs source and fate, highlight the main concepts of inflammasome activation via particulates, and explore recent advances in using inflammasome activation for assessment of MNP immunotoxicity. We also discuss the impact of co-exposure and MNP complex chemistry in potential inflammasome activation. Development of robust biological sensors is crucial in order to maximize global efforts to effectively address and mitigate risks that MNPs pose for human health.

BACKGROUND: The inflammatory responses are central components of diseases associated with particulate matter (PM) exposure, including systemic diseases such as cardiovascular diseases (CVDs). The aim of this study was to determine if exposure to PM, including respirable dust or quartz in the iron foundry environment mediates systemic inflammatory responses, focusing on the NLRP3 inflammasome and novel or established inflammatory markers of CVDs.

METHODS: The exposure to PM, including respirable dust, metals and quartz were determined in 40 foundry workers at two separate occasions per worker. In addition, blood samples were collected both pre-shift and post-shift and quantified for inflammatory markers. The respirable dust and quartz exposures were correlated to levels of inflammatory markers in blood using Pearson, Kendall τ and mixed model statistics. Analyzed inflammatory markers included: 1) general markers of inflammation, including interleukins, chemokines, acute phase proteins, and white blood cell counts, 2) novel or established inflammatory markers of CVD, such as growth/differentiation factor-15 (GDF-15), CD40 ligand, soluble suppressor of tumorigenesis 2 (sST2), intercellular/vascular adhesion molecule-1 (ICAM-1, VCAM-1), and myeloperoxidase (MPO), and 3) NLRP3 inflammasome-related markers, including interleukin (IL)-1β, IL-18, IL-1 receptor antagonist (IL-1Ra), and caspase-1 activity.

RESULTS: The average respirator adjusted exposure level to respirable dust and quartz for the 40 foundry workers included in the study was 0.65 and 0.020 mg/m3, respectively. Respirable quartz exposure correlated with several NLRP3 inflammasome-related markers, including plasma levels of IL-1β and IL-18, and several caspase-1 activity measures in monocytes, demonstrating a reverse relationship. Respirable dust exposure mainly correlated with non-inflammasome related markers like CXCL8 and sST2. CONCLUSIONS: The finding that NLRP3 inflammasome-related markers correlated with PM and quartz exposure suggest that this potent inflammatory cellular mechanism indeed is affected even at current exposure levels in Swedish iron foundries. The results highlight concerns regarding the safety of current exposure limits to respirable dust and quartz, and encourage continuous efforts to reduce exposure in dust and quartz exposed industries.

In this study, we present quantitative exposure-response data on silica exposure in male Swedish iron foundry workers receiving inpatient care for cardiovascular, cerebrovascular, and respiratory morbidity. The study show a significantly increased COPD risk at cumulative silica exposures that correspond to TWA silica below the Swedish OEL of 0.1 mg/m3. ObjectiveWe present quantitative exposure-response data on silica exposure in male Swedish iron foundry workers for cardiovascular, cerebrovascular, and respiratory morbidity.MethodsThis research is a cohort study of 2063 male Swedish iron foundry workers. From the Swedish National Patient Registers, data on morbidity incidence were retrieved. A historical measurement database of 1667 respirable silica exposure measurements from 10 Swedish iron foundries was used to calculate the cumulative exposure dose for each worker.ResultsIncreased morbidity risk for the whole group of foundry workers was determined for ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), bronchitis, and pneumonia. In addition, an increased risk for COPD at cumulative silica exposures ranging from 0.11 to 0.84 mg/m3 year is presented.ConclusionsThe study presents a significantly increased COPD risk at cumulative silica exposures below the Swedish occupational exposure limit.

INTRODUCTION: In light of potential negative health effects of cobalt exposure, a characterization of inflammatory mechanisms in exposed individuals is warranted. The current study investigated cobalt exposure in the Swedish hard metal industry and its relationship to inflammatory markers, including NLRP3 inflammasome activation and white blood cell (WBC) counts.

MATERIAL AND METHODS: Inhalable cobalt and dust exposures, and systemic cobalt levels, were determined for 72 workers in the hard metal industry and linear regression models were applied to correlate exposure to markers of inflammasome activation and WBC counts.

RESULTS: Mean exposures to inhalable dust (0.11 mg/m³) and cobalt (0.0034 mg/m³) were below the Swedish occupational exposure limits, and these low exposures did not correlate with any investigated outcomes. Instead, cobalt blood levels significantly correlated with a ca 10% decrease in IL-18 plasma levels per 10 nM cobalt increase. Furthermore, pre-shift cobalt blood and/or urine levels significantly correlated with some WBC measures, including decreased neutrophil-to-lymphocyte ratio, increased lymphocyte-to-monocyte ratio, and lymphocyte counts.

CONCLUSION: The low inhalable particle exposures had no impact on WBC counts and inflammasome activation. Instead, systemic cobalt levels, which also include skin exposure, demonstrated possible suppressive effects on inflammatory responses in cobalt-exposed individuals in the hard metal industry.

The formation of prostaglandin E2 (PGE2) is associated with adverse inflammatory effects. However, long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) comes with risk of severe side effects. Therefore, alternative ways to inhibit PGE2 are warranted. We have investigated the effects of tea extracts and the polyphenols epigallocatechin gallate (EGCG) and quercetin on PGE2 formation, determined by immunoassay, and protein expression, determined by immunoblotting, of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in human monocytes. Green and black tea extracts, and with a lower potency, Rooibos tea extract, inhibited lipopolysaccharide (LPS) and calcium ionophore-induced PGE2 formation. In addition, all tea extracts inhibited the LPS-induced expression of mPGES-1, and the green and black tea extracts also inhibited, to a lesser extent, COX-2 expression. The tea extracts only marginally reduced cPLA2 expression and had no effect on COX-1 expression. EGCG, present in green and black tea, and quercetin, present in all three teas, also inhibited PGE2 formation and expression of mPGES-1, COX-2 and cPLA2. Cell-based and cell-free assays were also performed to evaluate direct effects on the enzymatic activity of COX and PGE synthases. Mainly, the cell-free assay demonstrated partial inhibition by the tea extracts and polyphenols. However, the inhibition required higher doses compared to the effects demonstrated on protein expression. In conclusion, green and black tea, and to a lesser extent Rooibos tea, are potent inhibitors of PGE2 formation in human monocytes, and mediate their effects by inhibiting the expression of the enzymes responsible for PGE2 formation, especially mPGES-1.