Impact of Microplastic Exposure on Airway Inflammation in an Acute Asthma Murine Model
Article information
Abstract
Background
Widely distributed in the environment, microplastics (MPs) are increasingly recognized as potential respiratory hazards. While several studies suggest their role in worsening allergic airway diseases, findings remain inconsistent. This study aimed to investigate the immunologic effects of repeated MP exposure in an acute murine model of ovalbumin (OVA)-induced asthma.
Methods
Female BALB/c mice were assigned to four groups: control with vehicle, control with MPs, OVA-sensitized with vehicle, and OVA-sensitized with MPs. An acute asthma model was established by sensitizing and challenging mice with OVA. Spherical polystyrene MPs of 1−5 μm were administered intranasally at 300 μg daily from day 0 to 21. Lung inflammation was assessed via bronchoalveolar lavage fluid (BALF) analysis, histopathology, cytokine measurements, and macrophage polarization by immunofluorescence.
Results
MP exposure did not exacerbate allergic inflammation in OVA-sensitized mice. Instead, it led to reduced eosinophilic infiltration and lower levels of interleukin 5 (IL-5) and IL-13, compared to vehicle-treated OVA mice. In contrast, MP exposure in control mice increased tumor necrosis factor-α and decreased interferon-γ levels, upregulated epithelial alarmins (IL-25 and IL-33), and elevated inflammation scores. Alarmin levels, including IL-25 and IL-33, were elevated by MP exposure in control mice, whereas no significant differences were observed between vehicle- and MP-treated mice in the OVA-sensitized group. Macrophage analysis showed a shift toward M1 polarization only in control mice.
Conclusion
While MP exposure aggravated inflammatory responses in healthy lungs, it did not exacerbate airway inflammation in asthmatic mice.
Introduction
The global prevalence of plastics is driven by their strength, light weight, durability, chemical resistance, and affordability [1]. As of 2022, 400 million tons were produced worldwide, with production increasing by around 5 % annually [2,3]. Despite their utility, the chemical stability of plastics has led to their persistent accumulation in the natural environment.
Microplastics (MPs) are ubiquitous particles formed by the fragmentation of plastic waste, and are typically defined as synthetic polymer particles with a diameter of ≤5 mm [4,5]. These particles are increasingly detected across various environmental compartments, including air, water, and soil [5,6]. In particular, MPs are now recognized as airborne contaminants present in both indoor and outdoor environments, originating from sources such as textile abrasion, urban dust, industrial emissions, and the resuspension of settled particles [7]. They are transported by wind and deposited via atmospheric fallout, and detected even in remote regions, indicating their extensive environmental distribution. Furthermore, MPs have been detected in human biological samples, including lung tissue [8,9], sputum [10], urine [11], and blood samples [12], suggesting the possibility of systemic exposure. However, their hazardous effects on human health remain poorly understood.
Asthma is a chronic respiratory disease characterized by airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling [13], affecting around 300 million individuals globally, and contributing to approximately 1,000 deaths per day [14]. Environmental pollutants are recognized as key factors contributing to allergen sensitization, increased risk of exacerbation, and poor clinical outcomes [15]. Among airborne pollutants, MPs have recently been identified as potential detrimental factors influencing asthma outcomes in experimental studies [16-20]. However, a previous study reported conflicting findings, showing that airway resistance and Th2 cytokine levels did not differ significantly following MP exposure in a murine model of asthma [20].
In this study, we evaluated the impact of repeated MP exposure in an acute ovalbumin (OVA)-induced asthma murine model. We exposed OVA-sensitized mice and healthy controls to polystyrene MPs over the course of allergen challenge, and assessed resultant airway inflammation, cytokine profiles, and macrophage activation states.
Materials and Methods
1. Animals and experimental groups
Female BALB/c mice (6 weeks old; Orient Bio, Seongnam, Korea) were used in all experiments. The mice were randomly assigned to four experimental groups: (1) control treated with vehicle (CON/Veh, n=6), (2) control treated with MPs (CON/MP, n=8), (3) OVA-sensitized and treated with vehicle (OVA/Veh, n=8), and (4) OVA-sensitized and treated with MPs (OVA/MP, n=10). Each mouse was given a unique identification number, and a computer-generated randomization sequence using the RAND function in Excel was used to allocate animals to the respective groups, with equal probability of allocation to each experimental group.
2. Sensitization and antigen challenge protocol
Mice were sensitized with 25 µg of OVA (grade V, Sigma-Aldrich, St. Louis, MO, USA) combined with 1 mg of aluminum hydroxide (Sigma-Aldrich, Milwaukee, WI, USA) in 200 µL of phosphate-buffered saline (PBS), following the protocol described in our previous study (Figure 1) [21]. Twenty-four hours after the final OVA challenge, the animals were euthanized, and lung tissues, along with bronchoalveolar lavage fluid (BALF), were harvested for further analysis.
Experimental scheme of the mouse model of acute asthma and treatment with microplastics. OVA: ovalbumin; Alum: aluminum; s.c.: subcutaneous; i.n.: intranasal.
3. Microplastic exposure protocol
To evaluate the effects of MP exposure on both normal and OVA-induced asthmatic mice, fluorescent polystyrene MP spheres (FMG−Green Fluorescent Microspheres, 1.3 g/cc, 1−5 μm; Cospheric, Goleta, CA, USA) were suspended in PBS, and administered intranasally at a dose of 300 μg in 50 μL once daily from day 0 to 21 in the CON/MP and OVA/MP groups. Mice were lightly anesthetized with isoflurane, and held in an upright position (head upward) to ensure proper inhalation. The MP suspension was carefully instilled into the nostrils using a micropipette, and mice were maintained upright for an additional 2 minutes to facilitate inhalation. CON/Veh and OVA/Veh groups received the same volume of PBS without MPs, following the same procedure.
4. Bronchoalveolar lavage
BALF was collected at the endpoint of the experiment, as previously described. After euthanasia under anesthesia with an intraperitoneal injection of a Rompun and Zoletil mixture (1:4), BALF was collected. The exposed trachea was cannulated with silicone tubing attached to a 22-gauge needle on a 1 mL tuberculin syringe. BALF was collected after the instillation of 0.8 mL sterile PBS, and centrifuged at 3,000 rpm for 5 minutes at 4°C. The supernatants were collected and stored at −80°C. The bronchoalveolar lavage (BAL) cells were transferred onto glass microscope slides [21]. Slides were stained using the Diff–Quik staining kit (Sysmex, Kobe, Japan). Differential cell counts were performed for macrophages, neutrophils, lymphocytes, and eosinophils, with a total of 500 leukocytes counted from randomly selected fields under a light microscope.
5. Lung tissue histopathology
Lung tissues were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned at a thickness of 4 μm using a microtome. The paraffin sections were subsequently deparaffinized, and stained with hematoxylin and eosin (H&E) to assess airway inflammation. The degree of peribronchial inflammation was evaluated using a semi-quantitative scoring system based on the thickness of inflammatory cell infiltration around the airways [22].
6. Enzyme-linked immunosorbent assay
The concentrations of interleukin 5 (IL-5), IL-13, and tumor necrosis factor-α (TNF-α) in BALF were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s protocols (R&D Systems, Minneapolis, MN, USA). In addition, lung tissue homogenates were analyzed for levels of IL-17, interferon-γ (IFN-γ), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) using the same ELISA kits, except for IL-33, which was measured using a kit from Abcam (Cambridge, UK).
7. Lung macrophage immunofluorescence and polarization analysis
To investigate macrophage recruitment and polarization states, immunofluorescence (IF) staining was performed on lung sections to detect markers of M1 and M2 macrophage subsets. Paraffin-embedded lung sections (4 μm) were deparaffinized, subjected to heat-mediated antigen retrieval (pH 9.0 buffer, 121°C, 20 minutes), and blocked with serum. Sections were then incubated overnight at 4°C with primary antibodies against arginase-1 (Arg1; 1:50; Invitrogen, Carlsbad, CA, USA) as a marker of M2 macrophages, or inducible nitric oxide synthase (iNOS; 1:50; Abcam) as a marker of M1 macrophages. After washing, sections were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 647 (red) for both Arg1 and iNOS. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Stained sections were imaged using a confocal laser scanning microscope (Zeiss LSM 900, Zeiss, Oberkochen, Germany). Five randomly selected fields (400× magnification) per section were analyzed. Quantification of fluorescence intensity was performed using the Zeiss Zen Blue image analysis software. The number of iNOS-positive and Arg1-positive cells was manually counted, and the M1/M2 index was calculated as the ratio of iNOS⁺ to Arg1⁺ cells per field.
8. Statistical analysis
Data were analyzed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA, USA). All values are presented as the mean±standard error of the mean. Two-way analysis of variance (ANOVA) was performed to assess the main effects of OVA sensitization, MP exposure, and their interaction. When significant effects were detected, Tukey’s multiple comparisons test was applied for post hoc analysis. A p<0.05 was considered statistically significant. Key comparisons included the effect of MP in control mice (CON/MP vs. CON/Veh) and in OVA-challenged mice (OVA/MP vs. OVA/Veh), as well as the interaction effect between MP exposure and OVA sensitization.
9. Ethics statement
All procedures of animal research were provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent experiment provided by the Institutional Animal Care and Use Committee (IACUC) in the School of Medicine, The Catholic University of Korea (IACUC Approval number: CUMC-2023-0298-01).
Results
1. Microplastic exposure modulates inflammatory cell recruitment in BAL fluid
Total cell counts in BAL fluid were significantly increased in the OVA-sensitized groups, compared to controls (Figure 2A). MP exposure alone (CON/MP) did not significantly alter total BALF cellularity relative to vehicle-treated controls (CON/Veh), whereas OVA/MP mice showed a statistically significant reduction in total BAL fluid cell counts, compared to OVA/Veh (p<0.01).
Effects of microplastic (MP) exposure on bronchoalveolar lavage (BAL) fluid cellular composition. (A) Total BAL fluid cell counts. Differential cell counts of (B) macrophages, (C) eosinophils, (D) lymphocytes, and (E) neutrophils in control and ovalbumin (OVA)-sensitized mice treated with vehicle or MPs. Each symbol represents an individual mouse; open circles (○) indicate vehicle-treated mice, and closed circles (●) indicate MP-treated mice. *p<0.01; †p<0.001; ‡p<0.0001. ns: not significant.
Differential cell count analysis revealed that eosinophils and lymphocytes were markedly elevated in OVA/Veh mice, consistent with a Th2-high allergic response (Figure 2C, D). Moreover, MP exposure in OVA-sensitized mice (OVA/MP) resulted in reduced eosinophil and lymphocyte counts, compared to OVA/Veh (all p<0.01). MP exposure did not lead to significant changes in macrophage or neutrophil counts in either the control or OVA groups (Figure 2B, E). Supplementary Table S1 shows the inflammatory cell profiles for each mouse.
2. Microplastic exposure differentially affects airway inflammation in control and asthmatic mice
Histological examination using H&E staining revealed minimal peribronchial and perivascular inflammation in CON/Veh mice, while OVA-sensitized groups exhibited marked inflammatory cell infiltration (Figure 3). In addition, MP exposure in control mice (CON/MP) significantly increased airway inflammation, with higher inflammation scores than those observed in CON/Veh. Conversely, in OVA-sensitized mice, MP exposure (OVA/MP) resulted in reduced inflammatory cell infiltration and significantly lower inflammation scores, compared to OVA/Veh.
Histological assessment of airway inflammation following microplastic (MP) exposure. (A) Representative H&Estained (×20) lung sections from control and ovalbumin (OVA)-sensitized mice treated with vehicle or MPs (scale bar=50 μm). (B) Semi-quantitative inflammation scores based on peribronchial and perivascular inflammatory cell accumulation. Each symbol represents an individual mouse; open circles (○) indicate vehicle-treated mice, and closed circles (●) indicate MP-treated mice. *p<0.05; †p<0.0001.
3. Cytokine analysis reveals attenuation of Th2 responses by MP in OVA-induced asthma
ELISA revealed that levels of IL-5 and IL-13 in BAL fluid were significantly increased in OVA/Veh mice, compared to controls (Figure 4). However, these cytokines were reduced in OVA/MP mice, indicating that MP exposure may partially suppress Th2-driven responses. TNF-α levels were significantly increased in control mice following MP exposure (CON/MP vs. CON/Veh), whereas no significant differences were observed between OVA/Veh and OVA/MP groups.
Effects of microplastic (MP) exposure on cytokine and alarmin expression in bronchoalveolar lavage fluid (BALF) and lung tissue. BALF levels of (A) interleukin 13 (IL-13), (B) IL-5, and (C) tumor necrosis factor-α (TNF-α); lung homogenate levels of (D) interferon-γ (IFN-γ), (E) IL-17, (F) IL-25, (G) IL-33, and (H) thymic stromal lymphopoietin (TSLP). Each symbol represents an individual mouse; open circles (○) indicate vehicle-treated mice, and closed circles (●) indicate MP-treated mice. *p<0.05; †p<0.01; ‡p<0.001; §p<0.0001. ns: not significant; OVA: ovalbumin.
In lung tissue homogenates, IFN-γ levels were significantly reduced in MP-exposed control mice (CON/MP vs. CON/Veh), while no significant difference was observed between OVA/Veh and OVA/MP groups. In contrast, IL-17 levels were not significantly altered by MP exposure in either control or OVA-sensitized mice.
We analyzed the expression of epithelial alarmins, including IL-25, IL-33, and TSLP, in lung tissue homogenates. Following MP exposure, IL-25 and IL-33 levels were elevated in control mice (CON/MP vs. CON/Veh), whereas no significant differences were observed between OVA/Veh and OVA/MP mice. In contrast, TSLP levels were not altered by MP exposure in control mice, but were significantly reduced in the OVA/MP group, compared to OVA/Veh. Supplementary Table S1 shows the cytokine levels for each mouse.
4. Microplastic exposure selectively alters macrophage polarization in control mice
Lung sections were subjected to IF analysis to evaluate macrophage polarization using iNOS and Arg1 as markers of M1 and M2 phenotypes, respectively. Arg1 expression was generally higher in OVA-sensitized mice than in control mice, regardless of MP exposure, whereas iNOS expression did not differ significantly between OVA and control groups (Figure 5).
Effects of microplastic (MP) exposure on macrophage polarization in lung tissue. (A) Representative immunofluorescence images of arginase-1 (M2 marker, red), 4’,6-diamidino-2-phenylindole (DAPI; blue), and MP (green) staining. (B) Quantification of arginase-1 mean fluorescence intensity (MFI) in each group. (C) Representative immunofluorescence images of inducible nitric oxide synthase (iNOS; M1 marker, red), DAPI (blue), and MP (green) staining. (D) Quantification of iNOS MFI. (E) M1/M2 polarization ratio calculated as the ratio of iNOS to arginase-1 MFI. Each symbol represents an individual mouse; open circles (○) indicate vehicle-treated mice, and closed circles (●) indicate MP-treated mice.*p<0.05; †p<0.01; ‡p<0.0001. OVA: ovalbumin; ns: not significant.
In control mice, MP exposure (CON/MP) significantly increased the number of iNOS-positive (M1) cells compared to CON/Veh, while Arg1-positive (M2) cell numbers remained unchanged. As a result, the M1/M2 polarization index was significantly elevated in CON/MP relative to CON/Veh. In OVA-sensitized mice, MP exposure did not significantly affect the number of either iNOS- or Arg1-positive cells, and the M1/M2 index remained similar between OVA/MP and OVA/Veh groups.
Discussion
In this study, we investigated the immunologic effects of repeated MP exposure in a murine model of acute OVA-induced asthma. In contrast to our initial expectation that MP exposure would exacerbate allergic airway inflammation, we found that MP treatment did not intensify Th2-mediated responses in asthmatic mice. Instead, MP exposure resulted in a reduction of eosinophilic inflammation and Th2 cytokines (IL-5, IL-13) in OVA-sensitized animals. Interestingly, in healthy control mice, MP exposure increased pro-inflammatory cytokines, such as TNF-α and IFN-γ, elevated epithelial alarmins (IL-25 and IL-33), and induced histological signs of airway inflammation. However, these effects were not observed in OVA-challenged mice, suggesting a context-dependent immune modulation by MPs. Furthermore, macrophage polarization analysis revealed that MP exposure shifted the balance toward M1-like activation in control mice, but not in asthmatic lungs. These results imply that MPs exert differential immune effects, depending on the presence or absence of pre-existing airway inflammation.
Previous studies have shown contradictory results regarding the impact of MP exposure on airway inflammation in asthma models. Several factors may explain these discrepancies. First, particle size of the MPs may have contributed to the differential outcomes. Lu et al. [20] employed spherical polystyrene MPs of 1−5 μm, identical to those used in our study, and demonstrated no significant changes in airway inflammation, IL-4 or IL-5 levels, or airway resistance in a house dust mite (HDM)-induced asthma model, which corresponds to our study. In contrast, Han et al. [19] used significantly smaller MPs of 0.2 to 0.5 μm and reported increased airway inflammation, elevated IL-17 and IgE levels in OVA-sensitized asthma model, and further exacerbation of allergic responses when co-administered with di(2–ethylhexyl) phthalate (DEHP), a commonly used plasticizer. Furthermore, Fan et al. [23] conducted a particle size comparison study in rats using 100 nm to 2.5 μm MPs, and demonstrated that smaller particles showed greater pulmonary deposition, whereas 2.5 μm particles showed minimal lung accumulation, supporting these size-dependent effects. These findings collectively suggest that the relatively larger particle size used in our study may have limited the degree of airway penetration and cellular interaction, potentially explaining the attenuated or neutral inflammatory responses observed in our asthma model.
Furthermore, the route of MP administration may have influenced the observed outcomes. Both our study and that of Lu et al. [20], which reported similar findings, employed intranasal instillation of relatively large MPs of 1–5 μm. In contrast, studies reporting adverse effects of MP exposure often utilized the intratracheal route or whole-body inhalation system [23-26]. Given the larger particle size and intranasal delivery in our study, the extent of MP deposition in the lower airways may have been limited, potentially insufficient to induce or exacerbate airway inflammation.
The observed increase in epithelial alarmins such as IL-25 and IL-33 following MP exposure in control mice suggests that MPs can act as local stressors at the airway epithelial barrier [27,28]. Alarmins are typically released in response to epithelial damage or irritation, and their upregulation in naïve lungs implies that MPs are recognized as foreign irritants that are capable of triggering innate immune signaling [27,28]. In contrast, in OVA-sensitized mice, alarmin levels were generally elevated compared to controls, reflecting the ongoing epithelial stress and type 2 inflammation induced by allergen exposure. However, the expression of IL-25 and IL-33 did not differ significantly between OVA/Veh and OVA/MP groups, indicating that additional MP exposure did not further enhance alarmin signaling in already inflamed lungs.
Our macrophage polarization analysis also revealed a divergence in immune response, depending on the airway status. In control mice, MP exposure promoted a shift toward M1-like polarization, as evidenced by increased iNOS expression and a higher M1/M2 index, indicating an innate pro-inflammatory reaction [29]. However, this polarization shift was not observed in OVA-sensitized mice, where despite MP exposure, the M1/M2 balance remained unchanged. Interestingly, the observed reduction in Th2 responses occurred without a concurrent shift in the M1/M2 macrophage balance in the OVA-sensitized groups. Although M2 macrophages are known contributors to eosinophilic inflammation, this finding suggests that the immunomodulatory effects of MPs on allergic inflammation in our model are likely independent of this specific macrophage activation pathway. These data suggest that the impact of MPs on innate immune cell activation is diverse by the pre-existing immune environment of the lung.
This study has several limitations. First, we used an acute asthma model, which does not reflect the chronic features of asthma, such as airway remodeling. Second, we tested only one type and size of MP, using spherical polystyrene particles ranging 1 to 5 μm. Other particle sizes, shapes, or chemical compositions were not included, so our findings cannot be generalized to all types of MP exposure. Third, our study did not include an assessment of AHR, a key functional hallmark of asthma. Therefore, the direct impact of MP exposure on airway physiology remains to be determined. Fourth, the intranasal instillation method may have limited the effective dose of MPs reaching the lower airways, particularly in the mucus-rich environment of inflamed asthmatic lungs. It is also possible that a ‘ceiling effect’ in the robustly inflamed OVA model could have masked any potential additive pro-inflammatory effects of the MPs. Further studies using chronic models and a broader range of particle characteristics are needed.
In conclusion, our study demonstrates that repeated exposure to polystyrene MPs does not aggravate allergic airway inflammation in an acute murine asthma model. The immunologic effects of MPs appear to vary depending on the underlying inflammatory status of the airway, with distinct responses observed in healthy versus allergen-sensitized lungs. Further studies using chronic exposure models and a broader range of particle sizes, compositions, and shapes are needed to clarify their impact on asthmatic airways.
Notes
Authors’ Contributions
Conceptualization: Choi JY, Rhee CK. Methodology: Choi JY, Rhee CK. Formal analysis: Hur J. Data curation: Hur J. Software: Hur J. Validation: Hur J. Investigation: Choi JY, Hur J, and Rhee CK. Writing - original draft preparation: Choi JY. Writing - review and editing: all authors. Approval of final manuscript: all authors.
Conflicts of Interest
Chin Kook Rhee is a deputy editor, Yong Suk Jo is an editor, and Joon Young Choi is an early career editorial board member of the journal, but they were not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.
Funding
No funding to declare.
Supplementary Material
Supplementary material can be found in the journal homepage (http://www.e-trd.org).
Cytokine and inflammatory cell levels in individual mice.