Polystyrene nanoplastics disrupt the intestinal microenvironment by altering bacteria-host interactions through extracellular vesicle-delivered microRNAs

Abstract

Nanoplastics (NP) are emerging environmental pollutants with potential risks to human health. This study investigates how polystyrene-NP exposure disrupts the intestinal microenvironment and barrier function through bacteria-host interactions. Using in vivo models and bacterial sorting technology, we show that NP accumulation in the mouse intestine alters the expression of intestinal miR-501-3p and miR-700-5p, compromising tight junction protein ZO-1 and mucin (MUC)-13 expression, thereby increasing intestinal permeability. NP increases miR-98-3p, miR-548z, miR-548h-3o, miR-548d-3p, miR-548az-5p, miR-12136, and miR-101-3p levels in extracellular vesicles (EVs) derived from goblet-like cells, which can interfere with ZO-1 expression. NP also induces gut microbiota dysbiosis, characterized by elevated Ruminococcaceae abundance and altered EV characteristics from goblet cells. Lachnospiraceae internalize NP, and their EVs suppress MUC-13 expression. These findings reveal a mechanism by which NP compromises intestinal integrity and indirectly alters intestinal microbiota composition, potentially leading to adverse health outcomes.

Fig. 1. Evaluation of NP-induced side effects in mice.

A Distribution of NP (100 nm) in mice observed via in vivo imaging system (IVIS) for measuring fluorescence intensity (Near-infrared fluorescence). Effects of NP on (B) body weight gain, fat weight, and white adipose tissue. C intestinal length, and (D) serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) in mice orally administered with NP for 12 weeks. Data were shown as mean ± SEM (n = 12).

Fig. 2. The effects of NP on intestinal barrier function.

A Accumulation of NP in enterocyte-like differentiated Caco-2 cells after treatment for 24 and 48 h. Red stain: FluoSpheres nano-polystyrene (NP); Blue stain: DAPI. Scale bars indicate 100 µm. B Intestinal ZO-1 level in NP-treated mice for 12 weeks via IHC stain. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mean ± SEM (n = 12) (* p value < 0.05). C Enterocyte-like differentiated Caco-2 cells were treated with different concentrations of NP (10², 10⁴, 10⁶ particles/mL) for 48 h. Immunofluorescence staining of tight junction ZO-1 (green). Cell nuclei were counterstained by DAPI (blue). Scale bars indicate 20 µm. D The suppressions of ZO-1 and occludin in enterocyte-like differentiated Caco-2 cells with NP treatment measured by Western blot for concentration-dependent manner (upper) and time-dependent manner (below). E Intestinal leakage evaluation in enterocyte-like differentiated Caco-2 cells treated with NP for 48 h by measuring FITC-dextran permeability. Data were shown as mean ± SD (n = 3).

Fig. 3. Alterations in intestinal miRNA expression due to NP exposure and interferes ZO-1 expression.

A Principal components analysis (PCA) of miRNA sequencing results from colonic tissue of NP-treated mice (n = 6) for 12 weeks. B Heatmap and fold change of significantly different miRNAs post NP treatment (p < 0.05). C Gene Ontology (GO) annotations predicting gene expression changes following miRNA interference (GO0034329 for cell junction) in the colon. D Heatmap predicting miRNAs (miR-501-3p and miR-700-5p) potentially interfering with ZO-1. Validation of miR-501-3p and miR-700-5p interference with ZO-1 expression in enterocyte-like differentiated Caco-2 cells by (E) Immunofluorescence stain (Scale bars indicate 20 µm) and (F) Western blot. Data are presented as means ± SD (n = 3). G Schematic illustration of NP impact on ZO-1 expression and intestinal barrier function.

Fig. 4. Impact of NP on MUC-13 expression and intestinal miRNA interference in mice exposed for 12 weeks.

A Alcian blue-periodic acid Schiff (AB-PAS) staining of intestinal mucus in mice with or without NP treatment. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mean ± SEM (n = 12). B IHC stain of intestinal MUC-13 in NP-treated mice. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mean ± SEM (n = 12) (*** p value < 0.001). C MUC13 expression in NP-treated enterocyte-like differentiated Caco-2 cells for 48 h by ICC stain (Scale bars indicate 20 µm), qPCR, and Western blot. Data are presented as means ± SD (n = 3). Significant difference was shown by different letters (* p < 0.05; *** p < 0.001). D Western blot analysis of MUC-13 levels in goblet-like LS174T cells treated with NP for 48 h. E Heatmap predicting various intestinal miRNAs suppressing MUC-13 in NP-exposed mice. Validation of miR-700-5p interference on MUC-13 in enterocyte-like differentiated Caco-2 cells by (F) qPCR. G ICC stain (Scale bars indicate 20 µm), and (H) Western blot. Data are presented as means ± SD (n = 3). Significant difference was shown by different letters (***p < 0.001). I Schematic of NP impact on MUC-13 and mucus secretion in the gut.

Fig. 5. NP-induced changes in gut microbiota composition in mice.

A Venn diagram showing shared and unique ASVs of gut microbiota between the control and NP treatment groups. B β-diversity analysis of gut microbiota samples using principal coordinate analysis (PCoA) and Partial Least Squares Discriminant Analysis (PLS-DA). C α-diversity analysis of microbiota composition based on the Simpson index. The center line represents the median (50th percentile). The box represents the interquartile range (IQR) between the 25th percentile (Q1) and the 75th percentile (Q3). Whiskers extend to the smallest and largest values within 1.5 × IQR. Outliers are shown as individual points. Group Control: Min = 0.8835, Q1 = 0.9197, Median = 0.9311, Q3 = 0.9418, Max = 0.9489. Group NP: Min = 0.9170, Q1 = 0.9424, Median = 0.9428, Q3 = 0.9503, Max = 0.9557. D Relative abundance of the top 10 classifications for family and genus levels of gut microbiota. E Heatmap of the top 35 species at the family level. Group information is presented vertically, while species annotation is displayed horizontally. F Fluorescence microscopy showing NP uptake by fecal microbes in mice 6 h after administration. Bacterial DNA is stained with DRAQ5 (red), and fluoresbrite YG carboxylate microspheres represent NP (yellow-green). Scale bars indicate 2 µm. G Identification of gut microbes that have taken up NP using flow cytometric sorting and 16S rRNA sequencing. α-Diversity (Simpson index) was calculated using QIIME2, with group differences assessed using t-tests and Wilcoxon tests. β-Diversity was analyzed using UniFrac distance metrics and visualized through PCoA and PLS-DA plots. Statistical significance of β-diversity differences was assessed using t-tests and Wilcoxon tests. Taxa differences were analyzed using Welch’s t-test in STAMP and permutation tests in R’s metagenomeSeq package, with p-values adjusted using the Benjamini and Hochberg False Discovery Rate method. Data are presented as means ± SD (n = 6).

Fig. 6. Pathways of NP-induced gut microbiota changes.

A The effects of NP treatment (1 × 10¹⁰ particles/mL) on the growth of various lactic acid bacteria (L. paracasei, L. acidophilus, and P. acidiloctici), Lachnospiraceae sp. (TSD-26; ATCC), and Ruminococcaceae sp. (TSD-27; ATCC). B Schematic of experimental process by interactions between bacterial EV and cell-derived EV. C Impact of Lachnospiraceae sp.-derived EV without or with NP treatment (1 × 10¹⁰ particles/mL) for 18 h on the growth of different bacterial species (L. paracasei, L. acidophilus, P. acidiloctici, and Ruminococcaceae sp.). D The impact of Ruminococcaceae sp.-derived EV without or with NP treatment (1 × 10¹⁰ particles/mL) for 44 h on the growth of different bacterial species (L. paracasei, L. acidophilus, P. acidiloctici, and Lachnospiraceae sp.). E Impact of goblet-like LS174T cells without or with NP treatment (10⁶ particles/mL) for 48 h on the growth of different bacterial species (L. paracasei, L. acidophilus, P. acidiloctici, and Lachnospiraceae sp. and Ruminococcaceae sp.). F Western blot of MUC13 inhibition by Lachnospiraceae sp.-derived EV. Data were shown as mean ± SD (n = 3) (* p value < 0.05). G Schematic representation summarizing the proposed mechanisms of NP-induced modulation of gut microbiota via EV. NP are taken up by Lachnospiraceae, whose EV suppress MUC13 expression in goblet cells. Concurrently, NP-modified EV from goblet cells promote the growth of Ruminococcaceae, collectively contributing to gut microbiota imbalance and potential intestinal barrier dysfunction.