The molecular mechanisms of toxicity responses such as inflammation and ROS production to PP stimulation were investigated both in vivo and in vitro. Our results showed increased inflammatory cells, cytokines, and chemokines in the BALF of PP-instilled mice, alongside higher ROS production in the lung tissues, as compared to the VC group. Histopathological analysis of the lung tissue of PP-instilled mice revealed lung damage, including the infiltration of inflammatory cells in the perivascular/parenchymal space, alveolar epithelial hyperplasia, and foamy macrophage aggregates. In vitro investigation revealed depolarisation of the mitochondrial membrane potential, decreased ATP levels, and ROS production as a result of PP stimulation. PP was also found to induce the production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and cell death, with an increase in the levels of p-p38 and p-NF-κB protein observed both in vivo and in vitro. Interestingly, p38 and ROS inhibition regulated toxic responses such as inflammatory cytokines and cell death. These results suggest that PP stimulation might contribute to the pathogenesis of inflammation within the respiratory system via NF-κB signaling, which is associated with mitochondrial damage (Fig. 10).
With advancements in technology, the exposure to airborne microparticles such as PM in daily life has been increasing. Microparticles can form as a result of industrial processes, with other sources including traffic or road construction [23, 28–30]. Recent studies have reported that various plastics, including PP, PS, and PET were detected in the atmosphere in various forms (particles, fibres, and vinyl) [3–6]. Recent studies have reported that submillimetre microplastic fragments (PP, PET, PS, and polyvinyl chloride) have been detected in human lung tissue, with PP microplastics the most frequently observed (both in particulate and fibrous form) [11]. Interestingly, other studies have reported the detection of 12 different microplastic polymers in the lung tissue of live humans, with PP (23%), PET (18%), resin (15%), and PE (10%) the most abundant [10]. This exposure to persistent matter, including microplastics, can lead to respiratory diseases, and chronic exposure to persistent matter has been reported to cause cancer after 10 to 20 years [12, 31–34]. Several studies have reported the risks of inhalation exposure to humans, however, the toxicity mechanisms of microplastics in the respiratory system remain poorly understood.
Recent various studies have evaluated toxicologic effects of microplastic, hypothesizing that microplastic in the atmosphere might cause risk to the respiratory system by inhalation exposure in humans. Microplastics are known to induce lung inflammation by lung infiltration through the upper and lower airways as a result of inhalation exposure. Moreover, the inhalation of nanosized plastic can lead to bronchial epithelial injury by epithelial barrier infiltration [3, 12]. Recent studies investigating the effects of microplastics on lung epithelial cells have reported that PS microplastic exposure induces inflammatory cytokines and cytotoxicity owing to the cellular uptake of particles in A549 cells [26]. In other studies, the exposure of A549 cells to PS nano-plastics over 24 h led to the accumulation of the particles, which led to the production of inflammatory cytokines and oxidative stress, with ROS production and lipid peroxidation observed [35]. As mentioned above, PS has been investigated in several toxicology and physiology studies, as it finds usage in various products owing to its remarkable thermoplastic polymer properties [36]. However, investigations on the toxicity of PP, the primary material in disposable products, are scarce. With the increased usage of disposable products, PP microplastic has aroused concerns about possibile adverse effects in humans. Interestngly, recent studies have reported that PP was detected in body organs including the lung of humans [10, 11]. For this reason, toxicity studies on PP are necessary. Our results showed that ROS production, inflammatory cytokines, and cytotoxicity were significantly increased in PP-exposed A549 cells as compared to the control (Fig. 7), and alveolar epithelial hyperplasia and inflammatory cell infiltration were observed in the histopathological results of PP-instilled mice (Fig. 3). These results suggest that PP microplastics stimulation might contribute to the pulmonary toxic response through lung epithelium injury.
Our results showed that the number of inflammatory cells, including macrophages, neutrophils, and lymphocytes in the BALF of PP-instilled mice increased in a dose-dependent manner as compared to the VC. Neutrophils, which are the most common leukocytes and are essential first responders during the initial phases of inflammation, were predominant in the BALF of PP-instilled mice (Fig. 2a). The granulation and activation of neutrophils causes pulmonary inflammation via the release of various inflammatory cytokines and chemokines [37–39]. Especially, helper T (Th) cytokines play important roles in inflammatory responses in pulmonary diseases such as asthma and pulmonary fibrosis [40, 41]. Previous studies reported that IL-17 overexpression and neutrophil accumulation in BALF of mice with diesel exhaust particulates (DEP)-induced lung inflammation were observed and that the Th17 pathway might be involved in DEP-induced inflammation [23]. We investigated the gene expression patterns after PP exposure to reveal the molecular mechanism associated with PP-induced lung inflammation. We observed that the expressions of Th17 signaling pathway-associated genes including those encoding C-C motif chemokine ligand (CCL) 2, CCL12, CCL17, CXCL1, and CXCL5 were increased in the lung tissue of PP-instilled mice. We presume that, PP stimulation might have induced lung inflammation through the Th17 signaling pathway (data not shown). Recently, nanoparticles such as TiO2, CeO2, and ZnO have been reported to activate neutrophil degranulation, inducing inflammatory tissue injury [42–44]. PM-instilled mice have previously been shown to suffer from an increase in the number of neutrophils followed by the release of inflammatory cytokines and chemokines [45]. Interestingly, our results showed that inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, MCP-1, and CXCL1/KC in the BALF of PP-instilled mice increased in a dose-dependent manner, and the histopathological results of PP-instilled mice showed inflammatory cell infiltration (Fig. 2b-f, Fig. 3). These results indicate that PP microparticle stimulation may contribute to neutrophilic lung inflammation.
Oxidative stress and endoplasmic reticulum (ER) stress resulting from cellular organelle injury caused by exposure to environmental factors such as chemicals and pathogens can lead to various diseases including pulmonary diseases via abnormal inflammation and immune responses [19, 20, 46, 47]. Our results show that PP-exposed A549 cells had significantly increased ROS production and levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD)1, SOD2, and glutathione peroxidase-1 (GPX1). However, the protein levels of ER stress markers such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) were unchanged (Supplementary 1, 2). Interestingly, nuclear factor erythroid-2-related factor 2 (Nrf2) protein levels (total and nuclear) in PP-exposed A549 cells were significantly increased compared to the control, which might have regulated the expression of antioxidant proteins that protect against oxidative damage (Supplementary 3). Despite the need for more studies, we speculate that oxidative stress may be an indirect result of mitochondrial damage. Previous studies have reported that nanoparticles can damage mitochondria, and induce toxicity [27, 48–50]. Recent studies have reported that ultrafine dust exposure induced oxidative stress and mitochondrial damage in bronchial epithelial cells (BEAS-2B) and monocyte/macrophage cell lines (RAW 264.7 cells) [27]. NH2-PS stimulation induces mitochondrial dysfunction, leading to decreased ATP levels, DNA degradation, and a decline in the mitochondrial membrane potential of BEAS-2B and RAW 264.7 cells [48]. Our results showed that PP exposure induces mitochondrial dysfunctions such as mitochondrial depolarization and decreases ATP levels (Fig. 5). Interestingly, DRP1 proteins merged into the damaged mitochondrial regions (Fig. 6), which might be a quality control mechanism for preserving a healthy mitochondrial network via fission [51]. These results suggest that PP stimulation causes mitochondrial damage and that long-term PP microplastic exposure may potentially lead to mitochondrial diseases.
Recent studies have reported that airborne microplastics cause inflammation through various pathogenesis, such as dust overload, oxidative stress, and cytotoxicity [52–55]. In particular, ROS overproduction by particle exposure-induced inflammation and cytotoxicity is mediated by the release of cytokines and inflammatory mediators due to the translocation of nuclear factor NF-κB in cell signaling pathways [56–58]. Various studies have reported that oxidative stress causes NF-κB activation via the phosphorylation of MAPKs such as p38, ERK, and JNK, which regulate important cellular processes such as proliferation, stress responses, apoptosis, and immune defense [24, 25, 59–61]. Recent studies have reported that the persistent activation of p38 significantly contributes to the pathogenesis of Th2 low neutrophilic inflammation, which is associated with severe asthmatic phenotypes [62]. In addition, cigarette smoke-treated mice have shown p38 activation and release of pro-inflammatory cytokines and chemokines, which lead to neutrophilic lung inflammation. These findings are in agreement with the results obtained from chronic obstructive pulmonary disease (COPD) patients [63, 64]. In asthmatic patients, activated p38 MAPK contributed to TNF-α secretion from natural killer (NK) cells stimulated by IL-12, and the secretion of IL-6, IL-8, and MCP-1 were also partially dependent upon p38 activation [65, 66]. Interestingly, recent studies have reported that apoptosis was induced through p38 signaling by hydrogen peroxide, which is used as an oxidative stress inducer [67]. We demonstrated that PP stimulation caused inflammatory response, oxidative stress, and cell death. Interestingly, we also observed that the inflammatory cytokines and cell death induced by PP stimulation were reduced by p38 and ROS inhibitors (Fig. 9). These results suggest that lung neutrophilic inflammation via PP stimulation may be a potential therapeutic target that includes p38 inhibition.