Immunodysregulatory potentials of polyethylene or polytetrafluorethylene microplastics to mice subacutely exposed via intragastric intubation

Abstract

Microplastics (MPs) have been recently recognized as posing a risk to human health. The adverse health effects of MP exposure have been recently reported, especially via the oral exposure route. The present study investigated whether subacute (4 week) exposure to polyethylene (PE) or polytetrafluorethylene (PTFE) MPs via gastric intubation caused immunotoxicity. Two different sizes of PE MPs (6.2 or 27.2 μm) and PTFE MPs (6.0 or 30.5 μm) were administered to 6-week-old mice of both sexes at 0 (corn oil vehicle control), 500, 1000, or 2000 mg/kg/day (n = 4/group). No significant differences were observed between groups in the major thymic or splenic immune cell populations, including thymic CD4⁺, CD8⁺, CD4⁺/CD8⁺ T lymphocytes, and splenic helper T cells, cytotoxic T cells, and B cells. The ratio of interferon-gamma (IFNγ) to interleukin-4 (IL-4) in culture supernatants from polyclonally activated splenic mononuclear cells ex vivo (48 h) was dose-dependently decreased in female mice that received small- and large-size PTFE MPs. The IFNγ/IL-4 ratio was also decreased in the female mice dosed with large-size PE MPs. The serum IgG2a/IgG1 ratio was dose-dependently increased in male and female animals dosed with small-size PE MPs, in female animals dosed with large-size PTFE MPs, and in male animals dosed with small-size PTFE MPs. The present study implies that immune functions could be affected in animals exposed to MPs via gastric intubation. These effects are dependent on MP size, MP dose, MP polymer type, and mouse sex. Further investigations with longer exposure periods could be necessary to more clearly define the immunotoxic effects of MPs.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43188-023-00172-6.

Introduction

Humans can be exposed to microplastics (MPs) < 5 mm in diameter via ingestion, inhalation, or dermal absorption [1, 2]. MPs can be categorized into primary MPs present in food, beverages, household products, or personal care products, and secondary MPs generated through degradation of plastic products [2, 3]. Regardless of whether MPs originate from primary or secondary sources, MPs ≤ 20 μm have been reported to enter organs, and smaller MPs (1–10 μm) can cross cell membranes and the blood-brain barrier [3]. Furthermore, MPs can enter the circulation via the gut or lungs and can be translocated into distant organs including the heart, brain, kidney, reproductive organs, and immune system [1, 4].

Most studies of MP toxicity have been conducted in aquatic organisms, identifying various forms of toxicity such as decrease in growth or longevity, neurotoxicity, liver toxicity, and developmental toxicity [5, 6]. A few studies have evaluated MP toxicities in rodents, and MP exposure was reported to cause cytotoxicity and oxidative stress via direct cellular delivery, which could prompt inflammatory responses [7–9]. In addition, oral exposure to MPs in mice causes gut barrier dysfunction and disturbance of the gut microbiota [10, 11]. Maternal transfer of MPs from murine dams to their F1 offspring has also been reported, in which 10–45-µm polyethylene microplastics (PE MPs) were administered to dams via intratracheal instillation [12]. However, the study did not identify apparent biological changes or histopathological abnormalities in offspring. Specifically, neonatal serum triglycerides, acetylcholinesterase activity, and glutathione peroxidase activity were not significantly altered. Further, the effects of maternal MP delivery on tissue inflammation, necrosis, and lipid droplets in neonates were not examined in the brain, lung, heart, stomach, kidney, or intestines [12].

Several studies have demonstrated that MP exposure can adversely affect the immune system [1, 8]. Two pathways could be responsible for MP-mediated immune dysregulation, including direct interaction with cells at the port of MP entry, such as in phagocytic or antigen-presenting cells, and interactions in immune organs such as the spleen or lymph nodes following translocation via the lymphatic and/or blood circulatory systems [8, 13]. An in vitro study using human immune cell lines treated with 6 µm PE MPs demonstrated that production of proinflammatory interleukin-6 (IL-6) was decreased in both THP-1 cells with a dendritic cell phenotype and U937 cells with a macrophage phenotype [13]. Reactive oxygen species levels were increased in both cell lines, suggesting MPs could induce immune alterations by inflicting oxidative stress in immune cells. A few in vivo studies have reported MP immunotoxicity, primarily of polystyrene (PS) MPs, in the murine intestinal immune system [8, 9]. A recent study reported no apparent induction of inflammatory or oxidative stress in C57BL/6J mice orally exposed to 50 nm PS nanoplastics for 30 days, as demonstrated by unchanged mRNA levels of proinflammatory cytokines and oxidative stress-related inidcators in the liver, lung, and intestine, and unchanged serum levels of the proinflammatory cytokines [14].

PE and polytetrafluorethylene (PTFE) MPs are abundant contaminants in aquatic systems, food, and drinking water [15, 16]. Two prior studies have evaluated the immunomodulatory effects of PE MP exposure in rodents [17, 18]. These studies focused on PE MP toxicity in the gut immune system and second-generation PE MP immunotoxicity, and the findings indicated that PE MP immunotoxicity should be investigated in systemic innate and adaptive immunity. To the best of our knowledge, no prior studies have evaluated the immunotoxicity of PTFE MPs. Therefore, the present study was conducted to evaluate potential adverse effects of PE MPs and PTFE MPs on humoral and cell-mediated immunity in mice exposed to MPs for 4 weeks via intragastric administration. Two sizes of MPs with single- or double-digit µm diameters were used. Changes in the distribution of major thymic and splenic immune cells were examined, and serum levels of two major IgG isotypes (IgG1 and IgG2a) were also measured. In addition, production of interferon-gamma (IFNγ), interleukin (IL)-4 and IL-13 following ex vivo activation of splenocytes was quantified, as these cytokines are critical for driving type-1 and type-2 immune responses.

Materials and methods

Animals

Five-week-old male and female ICR mice purchased from KOATECH (Pyeongtaek, Korea) were acclimatized for 1 week prior to initiating experiments. Mice were housed in a specific pathogen-free animal facility at the Preclinical Research Center of Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), and the room was maintained at a room temperature 22 ± 1℃ with 50 ± 10% relative humidity and a 12 h light: dark cycle. All mice had ad libitum access to standard rodent food and autoclaved filtered water. All animal care and experimental procedures were conducted based on OECD TG 408: Repeated dose 90-day oral toxicity study in rodents [19] and were approved by the DGMIF IACUC (approval numbers DGMIF-20081404-01 & DGMIF-21031601-00).

Test substances and mouse exposure

Two different sized PE MPs and PTFE MPs were prepared: PE MPs: 6.2 ± 2.0 and 27.2 ± 7.9 µm, PTFE MPs: 6.0 ± 2.1 and 30.5 ± 10.5 µm. Single-digit average micrometer diameters (6.2 µm for PE MPs and 6.0 µm for PTFE MPs) are hereafter denoted as SM, and double-digit average micrometer diameters (27.2 µm for PE MPs and 30.5 µm for PTFE MPs) as DM. MP preparation was conducted as described previously [20, 21]. Briefly, 5 mm PE or PTFE beads were frozen in dry ice at − 78 °C followed by grinding with a homogenizer. The MPs were separated stepwise using 50 and 10 µm mesh filters and washed with 100% ethanol 5 times followed by drying at 50 °C for 48 h. Average MP particle size was determined using a particle size analyzer (ELS-Z2Plus, Otsuka Electronics, Japan), and particle shape was assessed by 3D profile using confocal microscopy (Keyence, Itasca, IL, USA) [20, 21]. Intragastric PE MP or PTFE MP administration was conducted in DGMIF. PE MPs or PTFE MPs were suspended in corn oil [22] for maintaining evenly dispersed MPs during preparation and administration (Daijung Chemicals & Metals, SiHeung, Korea) and administered once daily for 28 days at doses of 0 (vehicle), 500, 1000, and 2000 mg/kg. Four male and four female mice were assigned to each dose group. Concerning the IACUC recommendation for using a smaller number of mice, the number of 4 mice per gender to each dose group was calculated through Lamorte’s Power Calculations, which estimated sample sizes for p = 0.05 and 80% power level [23]. Since the approximate lethal dose for both size PE MPs or PTFE MPs was 2000 mg/kg or greater [20, 21], the present 4 week repeated oral dose study adopted 2000 mg/kg as high dose level.

Flow cytometric quantification of major splenic and thymic immune cells

CD4⁺ helper T, CD8⁺ cytotoxic T, and CD45R/B220 B lymphocyte populations were measured in single-cell splenocyte suspensions, and CD4⁺CD8⁺ immature T, CD4⁺ T, and CD8⁺ T lymphocyte populations were measured in single-cell thymocyte suspensions as described previously [Supplementary figure, 24]. Anti-CD4-fluorescein isothiocyanate (FITC) mAb (BD Biosciences, San Diego, CA, USA) and anti-CD8-phycoerythrin mAB (PE, BD Biosciences) were used to identify CD4⁺ or CD8⁺ T lymphocyte populations, respectively. Anti-CD3-FITC mAb (BD Biosciences) and anti-CD45R/B220-PE mAB (BD Biosciences) were used to identify B lymphocyte populations.

Ex vivo splenic lymphocyte cytokine production

Splenic single-cell suspensions (10⁶ cells/ml) were stimulated with immobilized anti-CD3 mAb (10 µg/10⁶ cells, BD Biosciences) and human recombinant IL-2 (10 U/10⁶ cells, Roche Korea, Seoul, Korea) for 48 h in a 5% CO₂ incubator. Cells were cultured in RPMI1640 medium (Biowhittaker, Walkersville, MD, USA) supplemented with 1 mM non-essential amino acids (Lonza Bioscience, Walkersville, MD, USA), 1 mM sodium pyruvate (Lonza Bioscience), 1% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine (Sigma-Aldrich), 50 µM 2-mercaptoethanol (Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA).

Measurement of serum immunoglobulins and culture supernatant cytokine levels

Serum was collected after centrifuging cardiac blood at 4000 rpm for 10 min at 4 ℃. Serum IgG1 and IgG2a were measured by sandwich Enzyme Linked Immunosorbent assays using goat anti-mouse IgG1 or IgG2a (Serotec, Oxford, UK) as the capture Abs and horseradish peroxidase-labeled goat anti-mouse IgG as the detection Ab (Sigma-Aldrich). IL-4, IL-13 and IFNγ levels in splenic culture supernatants were measured using OptEIA-paired capture and detection antibody sets (BD Bioscience) as described previously [24].

Statistical analyses

Statistical analyses were conducted using SigmaPlot version 14 (Systat Software, San Jose, CA, USA). Data were expressed as mean ± standard error of the mean (SEM) or mean ± standard deviation. Data normality were evaluated using a Shapiro-Wilk test. A Brown-Forsythe one-way analysis of variance (ANOVA) or Kruskal-Wallis ANOVA was performed for data with equal or unequal data distribution, respectively. When data were significantly different, a Student’s or Welch’s t-tests were further performed as post-hoc analyses to determine if relationships between two groups were statistically significant. P values < 0.05 were considered statistically significant.

Results

Unchanged body weight gain with oral MP administration

Body weight following repeated administration of exogenous substances has been adopted as an indicator for predicting gross adverse effects of substance exposure [24]. Body weight gain following 4-week administration of PE MPs did not significantly different between groups of different sex, MP dose, and MP size (Table 1). Similarly, administration of PTFE MPs did not affect body weight gain, except in male animals that received 500 mg/kg SM PTFE MPs, in which body weight gain was significantly decreased relative to vehicle control. Comparing the PE and the PTFE MPs administration, PTFE MPs groups resulted in less body weight gain than PE MPs groups regardless of gender. The in vivo experiments for PE MPs and PTFE MPs were undertaken with different batches of mice for one year interval, which could be a confounding factor for the difference in body weight gain.

Table 1.

Body weight gain (g) of mice exposed to MPs

MP size and dose (mg/kg/day)	Polyethylene		Polytetrafluorethylene
	Female	Male	Female	Male
Vehicle	8.40 ± 1.18	11.43 ± 1.36	5.12 ± 0.56	7.89 ± 2.19
SM 500	8.05 ± 0.99	11.58 ± 1.52	4.46 ± 1.54	3.68 ± 1.81*
SM 1000	7.75 ± 1.45	12.52 ± 1.25	2.07 ± 1.01	5.23 ± 2.29
SM 2000	8.33 ± 1.11	12.30 ± 1.21	3.62 ± 2.35	4.15 ± 1.16
DM 500	6.10 ± 0.79	10.30 ± 1.80	3.93 ± 1.79	6.15 ± 0.90
DM 1000	6.65 ± 1.47	9.90 ± 0.86	4.67 ± 0.90	5.79 ± 1.68
DM 2000	7.67 ± 0.79	9.92 ± 0.89	3.06 ± 0.99	6.07 ± 1.41

Body weight gain was calculated by subtracting the body weight at day 1 from the body weight at day 28 of MP administration. Vehicle control mice received corn oil. The administration volume was 10 ml/kg body weight. SM single-digit average particle size (PE 6.2 μm, PTFE 6.0 μm); DM double-digit average particle size (PE 27.2 μm, PFTE 30.5 μm). n = 4 mice/group. Data are expressed as means ± standard deviations

*p < 0.05 relative to vehicle control

Unchanged distributions of thymic and splenic lymphocyte populations

We examined the influence of PE MP and PTFE MP exposure on thymic immature and mature T lymphocyte and splenic T and B lymphocyte populations, as lymphocytes are core elements of humoral and cell-mediated immune functions. No significant differences were observed in thymic CD4⁺/CD8⁺, CD4⁺, or CD8⁺ T lymphocyte populations between groups of different sex, MP dose, and MP size (Table 2). Further, the proportions of splenic CD4⁺ T lymphocyte, CD8⁺ T lymphocyte, and B lymphocyte populations were not significantly different between groups (Table 3).

Table 2.

Distribution (%) of major thymic immune cell populations in mice exposed to MPs

MP size and dose (mg/kg/day)	PE		PTFE
	Female	Male	Female	Male
CD4+CD8+ T lymphocytes
Vehicle	78.1 ± 3.3	78.2 ± 4.1	83.0 ± 3.5	80.0 ± 3.5
SM 500	76.2 ± 1.2	81.9 ± 3.2	82.5 ± 2.8	82.0 ± 3.7
SM 1000	75.1 ± 2.4	76.2 ± 5.5	75.2 ± 7.2	79.5 ± 2.1
SM 2000	77.7 ± 4.1	79.1 ± 6.5	80.0 ± 4.2	81.6 ± 7.4
DM 500	75.1 ± 5.3	72.6 ± 14.3	81.2 ± 2.0	81.3 ± 3.7
DM 1000	77.8 ± 6.3	77.8 ± 1.6	82.4 ± 2.3	79.3 ± 4.3
DM 2000	78.6 ± 6.9	73.9 ± 4.3	82.9 ± 5.0	81.8 ± 2.1
CD4+ T lymphocytes
Vehicle	14.0 ± 3.1	13.2 ± 3.5	12.2 ± 3.4	15.1 ± 3.4
SM 500	15.2 ± 1.4	11.0 ± 2.7	12.9 ± 3.4	13.0 ± 3.5
SM 1000	14.7 ± 1.3	14.3 ± 3.8	16.6 ± 2.1	14.9 ± 2.5
SM 2000	14.0 ± 2.8	12.7 ± 4.3	15.3 ± 3.9	13.3 ± 6.0
DM 500	15.4 ± 4.1	16.7 ± 9.5	12.8 ± 2.4	13.7 ± 3.1
DM 1000	13.6 ± 3.9	13.5 ± 0.9	12.9 ± 1.9	14.9 ± 3.9
DM 2000	13.5 ± 4.4	16.6 ± 3.0	12.0 ± 2.8	12.9 ± 3.3
CD8+ T lymphocytes
Vehicle	3.6 ± 0.5	3.1 ± 0.7	3.1 ± 0.9	3.0 ± 0.3
SM 500	4.1 ± 1.2	3.1 ± 1.0	3.2 ± 1.1	3.3 ± 0.4
SM 1000	4.9 ± 1.3	4.6 ± 1.0	3.2 ± 0.9	3.7 ± 0.1
SM 2000	4.0 ± 1.6	3.5 ± 1.4	2.7 ± 0.6	2.9 ± 1.5
DM 500	4.6 ± 2.5	4.9 ± 3.5	3.7 ± 0.9	3.5 ± 0.8
DM 1000	3.9 ± 0.8	3.9 ± 1.1	2.8 ± 0.2	4.0 ± 1.3
DM 2000	3.2 ± 0.3	4.8 ± 0.6	3.5 ± 2.0	3.6 ± 1.0

Vehicle or MPs were administered to mice as described in Table 1. Proportions of thymic T lymphocyte subset were evaluated through fluorescence-activated cell sorting analysis. SM single-digit average particle size (PE 6.2 μm, PTFE 6.0 μm); DM, double-digit average particle size (PE 27.2 μm, PFTE 30.5 μm). n = 4 mice/group. Data are expressed as means ± standard deviations

Table 3.

Distribution (%) of major splenic immune cells in mice exposed to MPs

MP size and dose (mg/kg/day)	PE		PFTE
	Female	Male	Female	Male
CD4+ T lymphocyte
Vehicle	44.4 ± 11.1	45.1 ± 11.4	33.6 ± 1.1	32.0 ± 7.0
SM 500	39.0 ± 10.3	38.5 ± 8.6	29.4 ± 7.0	27.3 ± 3.7
SM 1000	34.3 ± 9.8	40.9 ± 10.4	30.3 ± 3.6	25.8 ± 11.3
SM 2000	41.9 ± 10.8	42.3 ± 9.7	35.0 ± 11.8	32.6 ± 6.9
DM 500	40.7 ± 8.2	42.4 ± 8.8	28.6 ± 7.5	26.9 ± 5.0
DM 1000	37.4 ± 14.3	42.9 ± 8.9	31.6 ± 2.5	27.5 ± 8.2
DM 2000	36.1 ± 6.2	42.5 ± 4.7	36.6 ± 2.4	27.9 ± 8.1
CD8+ T lymphocyte
Vehicle	15.1 ± 1.5	17.7 ± 2.0	10.8 ± 3.9	10.6 ± 1.4
SM 500	16.4 ± 1.5	14.7 ± 1.0	11.3 ± 2.2	11.7 ± 3.5
SM 1000	14.1 ± 3.5	16.6 ± 2.4	10.5 ± 2.3	10.7 ± 4.2
SM 2000	17.5 ± 7.1	17.4 ± 2.2	10.3 ± 3.3	13.3 ± 3.7
DM 500	17.6 ± 2.6	14.7 ± 3.8	11.1 ± 2.9	11.0 ± 2.1
DM 1000	12.6 ± 1.4	16.2 ± 2.7	11.5 ± 0.6	12.3 ± 3.6
DM 2000	17.3 ± 6.2	17.2 ± 3.5	11.4 ± 1.9	11.7 ± 2.7
B lymphocyte
Vehicle	25.7 ± 10.0	19.9 ± 7.6	17.4 ± 8.8	15.2 ± 2.2
SM 500	28.7 ± 10.6	31.1 ± 8.6	19.8 ± 8.9	18.2 ± 3.9
SM 1000	35.5 ± 13.1	25.6 ± 7.9	19.1 ± 4.3	19.1 ± 6.1
SM 2000	26.8 ± 16.0	26.4 ± 10.6	20.2 ± 5.4	14.1 ± 3.1
DM 500	25.1 ± 7.3	24.7 ± 7.4	21.1 ± 7.4	16.7 ± 3.1
DM 1000	35.8 ± 10.7	23.4 ± 6.7	17.9 ± 3.9	19.2 ± 5.9
DM 2000	31.0 ± 13.7	25.2 ± 3.6	17.7 ± 4.1	18.5 ± 4.7

Vehicle or MPs were administered as described in Table 1. Proportions of splenic lymphocyte subset were evaluated as described at Table 2. SM, single-digit average particle size (PE 6.2 μm, PTFE 6.0 μm); DM double-digit average particle size (PE 27.2 μm, PFTE 30.5 μm). n = 4 mice/group. Data are expressed as means ± standard deviations

Alterations in serum immunoglobulin and culture supernatant cytokine levels

IL-4 is a cytokine that induces isotype switching to IgG1, and IFNγ induces isotype switching to IgG2a in mice [24, 25]. Accordingly, the relative ratios of IgG2a/IgG1 and IFNγ/IL-4 have been adopted to determine the relative extent of the type-1 helper T cell (T_H1) response to the type-2 helper T cell (T_H2) response [24]. In addition, IL-13 is known to play a key role for driving toward T_H2 response [26]. Therefore IFNγ/IL-13 ratio could be adopted as another indicator for helper T cell related immune disturbance. The IgG2a/IgG1 ratio was not affected by MP dose, size, or polymer type (Fig. 1C and D). Nevertheless, the IgG2a/IgG1 ratio was increased in the female and male groups treated with SM-size PE MPs, although this trend did not reach statistical significance (Fig. 1C). The IgG2a/IgG1 ratios were higher in mice that received 2000 mg/kg SM PE MPs (female: 3.46 ± 0.72, male: 3.70 ± 1.20) than in the vehicle control (female: 2.37 ± 0.36, male: 2.48 ± 0.87). However, this trend was not present in mice that received DM PE MPs. Contrastingly, the IgG2a/IgG1 ratio was significantly lowered in the female 2000 mg/kg DM PE MP group (1.45 ± 0.46) relative to the female 500 mg/kg DM PE MP group (3.53 ± 0.42). The trend of dose-dependent IgG2a/IgG1 ratio increase was also present in the female DM-size PTFE groups and in the male SM-size PTFE groups (Fig. 1D).

Fig. 1.

Serum levels (mg/ml) of IgG1 or IgG2a in mice treated with polyethylene (PE) a or polytetrafluorethylene (PTFE) b microplastics (MPs). MPs or Vehicle were administered as described in Table 1. IgG2a/IgG1 ratios (PE-c, PTFE-d) were calculated by dividing IgG2a level by IgG1 level in each individual mouse. Data are expressed as mean ± SEM. SM, single-digit average particle size (PE 6.2 μm, PTFE 6.0 μm); DM double-digit average particle size (PE 27.2 μm, PFTE 30.5 μm). n = 4 mice/group. *p < 0.05 between the two groups

The IFNγ/IL-4 ratio was in general not affected by dose, size, or MP polymer type (Fig. 2C and D). However, the IFNγ/IL-4 ratio was dose-dependently decreased in the female SM-size and DM-size PTFE MP groups (Fig. 2D). This tendency was more robust in the female groups that received DM-size PTFE MPs, as the IFNγ/IL-4 ratio in the 2000 mg/kg group (14.9 ± 4.8) was significantly decreased compared with the vehicle control group (58.1 ± 18.5) and 500 mg/kg DM-size PTFE MP group (38.8 ± 8.6). Although it did not reach statistical significance, the trend of dose-dependent IFNγ/IL-4 ratio decrease was also present in female animals that received DM-size PE MPs (Fig. 2C). Contrastingly, the IFNγ/IL-4 ratio was significantly higher in male animals that received 2000 mg/kg DM-size PE MPs than in male animals that received 500 or 1000 mg/kg DM-size PE MPs. IL-4 level seems more influential on the ratio since dose-dependent IL-4 increases were observed in the groups with dose-dependent decrease in IFNγ/IL-4 ratio. For example, IL-4 levels versus vehicle control were 130.4, 139.2, 148.4% in the female 500, 1000, and 2000 mg/kg DM-size PTFE MP group, respectively, and 64.3, 75.6, and 113.0% in the female 500, 1000, and 2000 mg/kg DM-size PE MP group, respectively. Meanwhile, the relative IL-4 levels in the male 2000 mg/kg DM-size PE MP group was lower (62.8%) than in the male 500 (139.8%) or 1000 mg/kg (126.9%) DM-size PE MP group.

Fig. 2.

IFNγ level in splenic culture supernatants (ng/ml) in mice treated with PE (a) or PFTE (b) MPs. MPs or Vehicle were administered as described at Table 1. IFNγ/IL-4 ratios (PE-c, PTFE-d) were calculated by dividing IFNγ level by IL-4 level in each individual mouse. Data are expressed as mean ± SEM. SM, single-digit average particle size (PE 6.2 μm, PTFE 6.0 μm); DM double-digit average particle size (PE 27.2 μm, PFTE 30.5 μm). n = 4 mice/group. *p < 0.05 between the two groups

A similar trend was observed with the IFNγ/IL-13 ratio, in that the female group administered with 2000 mg/kg DM-size PTFE MPs demonstrated significantly (P < 0.05) lower (4.1 ± 1.1) IFNγ/IL-13 ratio compared with the vehicle control group (14.6 ± 3.2). Although no statistical significances were obtained, the IFNγ/IL-13 ratio was also lower in male mice administered with 2000 mg/kg DM-size PE MPs (10.3 ± 2.1) than the vehicle control group (24.9 ± 11.4). The IFNγ/IL-13 ratio was higher in male mice administered with 2000 mg/kg DM-size PE MPs (10.2 ± 3.8) than in male mice administered with 500 (6.2 ± 2.9) or 1000 mg/kg (3.4 ± 1.1) DM-size PE MPs, which trend was similar to the IFNγ/IL-4 ratio.

Discussion

PE is the most common plastic, and is used in packaging material, film, pipes and fittings, medical devices, toys, cosmetics, and other consumer products [27, 28]. PTFE, also known also as Teflon, is used in non-stick coatings, insulation material, film, lubricant, surgical graft material, cosmetics, and other consumer products [27, 29]. Dietary intake is a major route of human exposure to PE MPs and PTFE MPs [30]. Therefore, the present study aimed to evaluate the potential adverse effects of PE MPs and PTFE MPs on the overall immune responses in mice exposed to these materials for 4 weeks via daily intragastric administration of PE MPs or PFTE MPs. The present study, which was limited to relatively small numbers of experimental animals due to use of both sexes and multiple MP types and doses, demonstrated immunotoxic changes in humoral and cellular immunity, and immune alteration was influenced by MP type, size, dose, and mouse sex.

The immunomodulatory effects of in vivo PE MP and PTFE MP exposure have not been sufficiently addressed in rodent or human subjects. Although limited data are presently available, prior studies demonstrated that intragastric administration of 40–48 µm PE MPs induces subtle immune alterations, as ICR mice treated for 90 days with 3.75–60 mg/kg PE MPs exhibited a dose-dependent decreases in the proportions of mature splenic dendritic cells, but no differences in splenic T lymphocyte, B lymphocyte, or natural killer cell proportions [18]. The same study also reported increased serum IgA in mice exposed to PE MPs relative to vehicle control, but IgG, IgM, and IgE levels were unchanged by PE MP administration. The present study, in which ICR mice were treated with higher doses (500, 1000, 2000 mg/kg) of PE MPs for a shorter period (28 days), also did not identify significant differences in the distributions of splenic helper T cells, cytotoxic T cells, or B cells, nor in thymic helper T cells, cytotoxic T cells, or immature T cells. A significant change in the proportions of lymphocyte subpopulations can be induced through persistent, longitudinal, and profound insults of pathogenic microorganisms or toxic agents [31, 32]. Therefore, it could be premature to conclude that PE MPs do not affect peripheral major immune component cells in any context until longer periods of exposure, for example 90 days, have been tested. This interpretation could be also applied to the present observation that PFTE MPs did not affect the proportions of splenic or thymic lymphocyte subpopulations. Considering the deposit of average 27 µm size PE MPs in the lung, gastrointestinal system, and serum of mice orally administered for 4 week [20] but no detection of average 30 µm size PTFE MPs in the serum of mice orally administered once [21], duration of exposure will be a critical factor for accumulation or dissemination of MPs into various organs including immune organs, which could exert certain influences on immune function.

A robust dose-dependent decrease in the IFNγ/IL-4 ratio was present in ex vivo stimulated splenocytes from female mice treated with SM-size and DM-size PTFE MPs. However, the IFNγ/IL-4 ratio was significantly increased in ex vivo stimulated splenocytes from male mice treated with the highest dose (2000 mg/kg) of DM-size PE MPs relative to splenocytes from male mice treated with lower doses of DM-size PE MPs. Because the IFNγ/IL-4 ratio is considered to reflect the predominance of the type-1 anti-tumor or anti-viral immune response over the type-2 atopic immune response [24], the present findings that decrease of the IFNγ/IL-4 ratio by PFTE MP exposure could indicate a potential of skew toward type-2 over type-1 response following PTFE MP exposure. This tendency was further substantiated with decrease of IFNγ/IL-13 ratio by PTFE MP exposure. Meanwhile, this relationship was not present in mice exposed to PE MPs, implying that induction of T_H1 or T_H2 predominance could be dependent on animal sex, MP size, or polymer type. Skewing of the immune response to type-2 response frequently occurs following exposure to environmental or occupational toxicants [33, 34]. Previous investigations of the immunotoxic effects of MP exposure have been primarily focused on inflammation, with no conclusive findings regarding the MP-mediated inflammatory response [14, 17]. When C57BL/6J mice were exposed to 10–150 µm PE MPs at doses of 6, 60, or 600 µg/day for 5 weeks via chow administration, serum levels of the proinflammatory cytokine IL-1α were increased in all groups fed with PE MPs, and histopathological signs of intestinal inflammation were present [17]. Contrastingly, no obvious induction of the inflammatory response was reported in C57BL/6J mice intragastrically exposed to 50 nm PS nanoplastics for 30 days, as intestinal and circulating IL-1β, IL-6, and tumor necrosis factor-α were unchanged [14]. Overall, the immunoregulatory effects of MPs could be influenced by multiple confounding factors such as polymer type, particle size, exposure duration, and immune organs examined.

IgG isotype switching is regulated by cytokines secreted by activated T cells in the tissue microenvironment [35, 36]. IL-4 is the major cytokine that induces isotype switching to IgG1 or IgE, and IFNγ is the major inducer of switching to IgG2a in mice [24, 25]. This paradigm was observed in the present study, as in female animals exposed to DM-size MPs, the serum IgG2a/IgG1 ratio was concurrently decreased with the IFNγ/IL-4 ratio in ex vivo activated T cells. By contrast, in female animals exposed to DM-size PTFE MPs, the serum IgG2a/IgG1 ratio was increased, while the IFNγ/IL-4 ratio in ex vivo activated T cells was decreased. Considering reports demonstrating IFNγ-independent IgG2a production [37] and IL-4 independent IgG1 switching [38] in mice, further investigation of these regulatory mechanisms is necessary to interpret these seemingly conflicting findings. Measuring expression of the T-box transcription factor T-bet could be valuable, as T-bet regulates T cell-independent IgG2a switching [39]. IL-21 is a candidate cytokine that should be examined, as IL-21 potentiates IgG1 switching in the context of limited IL-4 [40].

In conclusion, although no significant changes were resulted on hematological parameters including major immune cell proportions and weight of immune organs including spleen and thymus [20, 21], our results demonstrated that PE MPs and PTFE MPs administered subacutely via intragastric intubation could disturb immune homeostasis, leading to an imbalance between type-1 and type-2 immunity. However, no toxicity was observed in the proportions of splenic and thymic lymphocyte populations. A future study with a longer administration period, for example 90 days of repetitive administration, could further delineate the immunomodulatory and immunotoxic effects of PE MP and PTFE MP exposure.

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Funding

This research was supported by the Korea Ministry of Environment (grant # 2020003120002) and the educational training program for the management of information on the hazards and risk of chemical substances funded by the Korea Ministry of Environment (entrusted to the Korea Chemicals Management Association).

Declarations

Conflict of interest

The authors have no conflict of interest to disclose.