Polystyrene microplastic particles induce endothelial activation.

Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the μm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.

Introduction

Since the last decades, living beings are increasingly faced with several novel environmental risk factors, such as particulate matter, plasticizers and microplastics. The latter are small synthetic and solid particles with a size ranging from less than 1 μm to 5 mm and can be divided in primary microplastics produced for instance for cosmetic and medical products and in secondary microplastics resulting from natural erosion and decomposition of macroplastics [1]. An example for the latter is polystyrene (PS) being present in particulate matter because of tire abrasion. Microplastics continuously accumulate in our environment due to the globally growing production of plastic reaching 368 million tons in 2019 according to the association of plastics manufacturers [2]. There is no doubt that plastic is a material of great benefit at relatively low production cost and offers a wide range of applications. Hence, the collective term plastic includes various polymers with polypropylene, polyethylene and polystyrene being the most widely used representatives in everyday life in regard to food packaging, disposable plastic or tire abrasion [2]. Microplastics have been detected in coastal regions, oceans and also in marine organisms like phytoplankton, mussels and fish, thus ultimately entering the human food chain [3-5]. Accordingly, a study tested human stool samples of different regions around the world positive for major microplastic polymers such as PS particles [6]. Of note, just recently the same group detected microplastic particles in human placenta and meconium as well [7]. Therefore, microplastics represent a novel environmental factor; however, their effects on human health are largely unknown. To date, only few in vivo studies investigated microplastics in this regard. Recent studies reported microplastic particle accumulation in different organs like liver and heart upon oral administration [8-10], demonstrating their uptake via the intestine into the circulation where they may interact with immune cells and the endothelium promoting inflammatory effects [11]. Additionally, PS particles have been shown to impair lipid metabolism in murine macrophages [12] and to trigger hepatoxicity [13]. In the context of a potential use of PS particles as drug carriers, Barshtein et al. observed elevated aggregation and endothelial adhesion of red blood cells treated with PS in vitro [14]. However, effects on the vasculature are largely unknown. For the first time, we show the capability of carboxylated PS particles to activate endothelial cells and enhance monocyte adhesion. In addition, we demonstrate their uptake by neutrophils in the blood.

Materials and methods

Cell culture and treatment

For in vitro experiments, we used murine cell lines: endothelial MyEND cells (myocardial endothelial cells, RRID: CVCL_2131) which we recently characterized in regard to endothelial markers and properties [15] as well as monocytic J774A.1 cells (CVCL_0358), which were cultured in a humidified incubator at 37°C and 5% CO2. MyEND cells were cultured using Dulbecco´s Modified Eagle´s Medium (DMEM, Gibco, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, PAN-Biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (PenStrep, 100 U/mL and 100 mg/mL, Sigma-Aldrich, Seelze, Germany). For J774A.1, DMEM GlutaMAX (Gibco) supplemented with 10% FCS and 1% PenStrep was used. PS particles conjugated with tetramethylrhodamine isothiocyanate (TRITC) or unconjugated (1 μm, carboxylated, Kisker Biotech, Steinfurt, Germany,) were used as a representative of microplastics abundantly occurring in our environment. Polystyrene as polymer component of the particles was confirmed by Raman spectroscopy (S1A and S1B Fig) and size distribution of the PS particles were measured by dynamic light scattering (1108±185.6 nm, Zetasizer Nano ZS90, Malvern Pananalytical, Malvern, UK) (S1C Fig). Using the above mentioned medium supplemented with 1% FCS, J774A.1 (sub-confluent) and MyEND (confluent) cells were starved for 2 hours and subsequently stimulated with PS particles (103, 105 and 107 particles/mL, which corresponds to 0.54 ng/mL, 54 ng/mL and 5.4 μg/mL) for 3 and 6 hours in a 12-well plate, respectively. RNA and supernatant were used for further real-time PCR and ELISA analysis.

Cell viability assay

Cell viability of J774A.1 and MyEND cells after 16 hours of exposure to PS particles was analyzed using the alamarBlue Cell Viability Assay (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer´s instructions. Both cell types were exposed to an increasing concentration of PS. Triton was used as positive control.

Animal experiments

Wild type C57BL/6N mice at the age of 10–12 weeks were given 2.5 mg fluorescent PS-TRITC particles (1 μm, carboxylated, Kisker Biotech) or PBS (up to 9 mice per group) as control by intravenous injection. All animal experiments were approved by the regional council Giessen (99/2019) and conform to the guidelines from directive 2010/63/EU of the European Parliament. After 3 hours, mice were anesthetized using Ketamin and Xylazin (120 mg and 12 mg/kg bodyweight) and euthanized by puncturing the left ventricle. Peripheral blood, liver and aortic tissue (aortic arch and thoracoabdominal aorta) were collected. Blood and liver samples were used for immunohistochemistry, aortic tissue for gene expression analysis.

Real-time PCR

For the analysis of mRNA expression, total RNA from aortic and liver tissue, J774A.1 and MyEND cells was isolated using RNA-Solv® Reagent (Omega Bio-tek, Norcross, GA, USA) following manufacturer´s instructions and reverse-transcribed with SuperScript reverse transcriptase, oligo(dT) primers (Thermo Fisher Scientific) and deoxynucleoside triphosphates (Promega, Madison, WI, USA). Real-time PCR was performed in duplicate in a total volume of 20 μL using Power SYBR Green PCR master mixture (Thermo Fisher Scientific) on a Step One Plus real-time PCR system in 96-well PCR plates (Applied Biosystems, Waltham, MA, USA). SYBR Green emissions were monitored after each cycle. For normalization, expression of Gapdh was determined in duplicates. Relative gene expression was calculated by using the 2-ΔΔCt and 2-ΔCt method for matched and independent pairs sampling respectively. Real-time PCR primers for mouse Il-1β, Tnf-α, Vcam-1, Icam-1, Saa1, Saa2, Saa3 and Gapdh were obtained from Microsynth AG (Balgach, Switzerland): Il-1β-F: 5’-GCC ACC TTT TGA CAG TGA TGA G-3’, , Tnf-α-R: 5‘-GTA GCC CAC GTC GTA GCA AAC-3’, Vcam-1-F: 5‘-TCT TAC CTG TGC GCT GTG AC-3’, Vcam-1-R: 5‘-ACC TAG CGA GGC AAA CAA GA-3’, Icam-1-F: 5‘-CAC GTG CTG TAT GGT CCT CG-3’, Icam-1-R: 5‘-TAG GAG ATG GGT TCC CCC AG-3’, Saa1-F: 5‘-CCC AGG AGA CAC CAG GAT GA-3’, Saa1-R: 5‘-TCA TGT CAG TGT AGG CTC GC-3’, Saa2-F: 5‘-TGC TGA GAA AAT CAG TGA TGC AA-3’, Saa2-R: 5‘-CCC AAC ACA GCC TTC TGA AC-3’, Saa3-F: 5‘-GAA AGA AGC TGG TCA AGG GTC-3’, Saa3-R: 5‘-TCC GGG CAG CAT CAT AGT TC-3’, Gapdh-F: 5‘-GTC TCC TGC GAC TTC AGC-3’, Gapdh-R: 5‘-TCA TTG TCA TAC CAG GAA ATG AGC-3’.

Enzyme-linked immunosorbent assay (ELISA)

Supernatant of J774A.1 and MyEND cells were analyzed for TNF-α and sVCAM-1 respectively, using mouse-specific ELISA (R&D Systems, Minneapolis, MN, USA) according to manufacturer’s protocol with the help of an Infinite M200 PRO plate reader (TECAN Instruments, Waltham, MA, USA).

Adhesion assay

Adhesion under static conditions

MyEND cells were grown to complete confluence in a 24-well plate. Cells were stimulated in DMEM supplemented with 1% FCS for 16 hours with PS (107 particles/mL). Untreated cells cultured in DMEM supplemented with 1% FCS served as control. J774A.1 cells were labelled with 1 μM of Calcein AM (eBioscience, San Diego, CA, USA, excitation: 495 nm, emission: 515 nm). After stimulation, MyEND cells were washed twice with 500 μL DMEM and 0.5x106 labelled J774A.1 cells were added per well and incubated for 1 hour in 5% CO2 at 37°C. After incubation each well was washed three times with 500 μL DMEM and 10 high power field digital images were taken using Axio Vert.A1 microscope with an AxioCam MRm camera (Carl Zeiss, Oberkochen, Germany). Adhered cells per HPF were counted using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Adhesion under flow conditions

For flow based adhesion assay, 5x105 MyEND cells were plated in μ-Slide VI0.4flow chamber (ibidi, Martinsried, Germany) in DMEM supplemented with 10% FCS and 1% PenStrep and grown to complete confluence. Cells were stimulated as described above. After stimulation, μ-Slide was connected to a 20 cm silicon tubing attached to a luer lock adaptor, which was in turn connected to a 50 mL syringe attached to a Perfusor VII pump (B Braun AG, Melsungen, Germany). For flow conditions, a flow rate of 0.53 mL/min (which corresponds to laminar flow of 0.5 dyne/cm2) was maintained. Endothelial layer was perfused with DMEM for 2 min to remove any debris and dead cells. After washing, Calcein-labeled J774A.1 cells at a concentration of 1x106 cells/mL were perfused for 2 hours at a constant shear stress of 0.5 dyne/cm2, followed by perfusion with DMEM for 30 min to remove unbound cells. The last 2 min of washing were recorded by a DM550B fluorescence microscope with a DFC300FX camera (Leica Microsystems, Wetzlar, Germany) and 20 HPF digital images from this recording were subsequently used for the analysis. Adhered cells per high power field were counted using ImageJ software.

Immunohistochemistry and imaging

Liver tissue and peripheral blood from wild type mice administered with PS-TRITC or PBS was collected and erythrocytes were lysed. Blood smear samples and liver cryosections were prepared and PS-TRITC particles were subsequently observed using an Axio Vert.A1 microscope with an AxioCam MRm camera (Carl Zeiss). Blood smear samples were stained for DAPI and rat anti-mouse Ly6G (Invitrogen, Waltham, MA, USA). Goat anti-rat IgG conjugated to FITC (Invitrogen) was used as secondary antibody. Liver sections were stained for DAPI and Alexa Fluor 488-WGA (Thermo Fisher Scientific). Confocal images were acquired on a Leica TCS SP2 microscope. Image analysis was performed using Leica LAS AF and ImageJ.

Statistical analysis

All data are represented as means ± SD. Groups were compared using parametric 2-tailed Student t-test (GraphPad Prism, version 9.01; GraphPad Software, La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant. Real-time PCR was performed in technical duplicates.

Results

In initially performed studies assessing cell viability using both murine monocyte/macrophage J774A.1 cells and endothelial MyEND cells, PS particles with a concentration ranging from 103−107 particles/mL did not cause any cytotoxic effects in these cells after 16 hours, the longest period of stimulation with PS particles in this study (Fig 1A). We performed pilot studies in order to define time- and dose-dependence for both cell lines and observed greatest effects on inflammatory cytokine expression with a stimulation period of 3 hours for J774A.1 and 6 hours for MyEND cells, respectively using 107 particles/mL (S2A Fig). At this concentration, a monolayer of MyEND cells is sparsely covered with single, doublets or small aggregates of PS particles ensuring appropriate particle/cell contact under our experimental conditions (S2B Fig). In J774A.1 cells, PS particles induced the expression of inflammatory cytokines such as Il1-β and Tnf-α as well as TNF-α release. In endothelial MyEND cells, PS particles up-regulated the expression of the major endothelial adhesion molecules vascular cell adhesion molecule (Vcam)-1 and intercellular adhesion molecule (Icam)-1 indicative of endothelial activation. Moreover, stimulation with PS particles increased sVCAM-1 release from endothelial cells (Fig 1B).

Fig 1

Effects of PS particles on cell viability and inflammatory gene expression.

(A) Cell viability (%) of J774A.1 and MyEND cells after 16 hours of PS particles stimulation (103−107 particles/mL) was determined using alamarBlue cell viability assay, n = 3–5. (B) Il1-β, Tnf-α expression and TNF-α levels after 3 hours of PS particles stimulation (107 particles/mL) in J774A.1 cells (above) and Vcam-1, Icam-1 expression and sVCAM-1 levels after 6 hours of PS stimulation (107 particles/mL) in MyEND cells (below) were determined by real-time PCR and by ELISA, respectively, n = 7–10. *P<0.05, **P<0.01, ***P<0.001 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

To investigate a potential functional effect of PS particles on monocyte adhesion under static and flow (0.53 mL/min) conditions, we incubated confluent MyEND cells with PS particles for 16 hours to allow adequate expression of the adhesion molecules at the protein level on the cell surface. Subsequently, we added Calcein-AM-labelled J774A.1 cells, followed by a standardized washing protocol. Following PS particle stimulation, significantly more adherent monocytic J774A.1 cells were detected under static as well as under flow conditions by fluorescence microscopy (Fig 2A and 2B).

Fig 2

Effects of PS particles on monocytic cell adhesion to endothelial cells.

Fluorescence microscopy images depicting Calcein-AM-labelled J774A.1 cells on a MyEND monolayer after stimulation with PS particles for 16 hours (107 particles/mL), one hour adhesion under static (A) and two hours under flow (B) conditions and subsequent washing. Scale bars = 100 μm. Adherent cells per high power field (HPF) were quantified. N = 4–6. **P<0.01 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

For in vivo studies, C57BL/6N wild type mice were administered with 4.65x109 TRITC-conjugated PS particles (corresponds to 2.5 mg, 1 μm, Kisker Biotech). PS particles were consequently detected in the liver by confocal microscopy (Fig 3A) and significantly up-regulated the hepatic expression of serumamyloid A (Saa)1, Saa2 and Saa3 (Fig 3B), indicating an inflammatory acute-phase response of the liver to PS particles [16].

Fig 3

Effects of PS particles in vivo: Hepatic accumulation and mRNA expression.

(A) Confocal microscopy composite of liver sections from mice 3 hours after injection with 2.5 mg TRITC-conjugated PS particles. blue: DAPI, red: Alexa Fluor 488-WGA, green: TRITC-PS (appear intracellularly in yellow). Scale bars: overview = 50 μm, insertion = 10 μm, z-stack in right panel = 5 μm. (B) Hepatic Saa1, Saa2 and Saa3 expression of mice administered with either 2.5 mg TRITC-conjugated PS particles or PBS as control were determined by real-time PCR, n = 8–9 mice/group. **P<0.01, ***P<0.001 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

To characterize potential interactions of PS particles with circulating leukocytes, blood smear samples were prepared after 3 hours of administration. Free PS particles as well as accumulated PS particles taken up by Ly6G-positive cells were detected in the peripheral blood, identifying neutrophils as a particles-clearing blood leukocyte population (Fig 4A). In line with our findings in vitro, we determined significantly enhanced levels of the cytokine Il-1β as well as the endothelial activation markers Vcam-1 and a trend (P = 0.069) towards elevated Icam-1 expression in the aortic tissue from mice injected with PS particles (Fig 4B).

Fig 4

Effects of PS particles in vivo: Peripheral blood and aortic tissue.

(A) Overlay of brightfield and epiflourescence (left) and rendered confocal image stack (right) of peripheral blood cells 3 hours after administration of 2.5 mg TRITC-conjugated PS particles. Whole blood was collected and red blood cells were lysed prior to staining. Scale bar = 10 μm. Blue: DAPI, red: FITC-Ly6G, green: TRITC-PS. (B) Il-1β, Vcam-1 and Icam-1 expression in aortic tissue of mice administered with either 2.5 mg TRITC-conjugated PS particles or PBS as control were determined by real-time PCR, n = 7 mice/group. *P<0.05, **P<0.01 vs. control (con). Data were analyzed by Student t-test and are depicted as mean±SD.

Discussion

Microplastic pollution is an increasing global challenge that raises concerns about our health as well. First studies show the translocation of these particles across the intestinal barrier into the organism. What consequences on biological processes this uptake may have and whether the particles thus initiate pathological processes is largely unknown. Only few studies investigated the effects of mainly polystyrene so far, for instance showing cytotoxic effects on the gut barrier function [17] and lung epithelial cells [18] in vitro. Performing a long-term exposure of the marine organism daphnia magna to polystyrene microplastic, Trotter et al. recently showed an impact on morphology, number of offspring and the organisms´ proteome, suggesting an altered supply for nutrients and overall fitness [19]. Using the same model organism, Kelpsiene et al. reported a reduced life-time upon exposure to polystyrene nanoplastics [20]. Of note, some studies reported translocation in a surface charge-dependent manner in both marine organisms and rats [8, 21]. Based on these observations and the fact that due to weathering processes and subsequent surface oxidation, microplastic particles are expected to acquire carboxylic groups (-COOH) and hence are negatively charged [22], we therefore have used carboxylated PS particles in our study as well.

We are aware that our study has some limitations, e.g. regarding dosage and route of exposure. Until today, there are only speculations about the actual exposure and uptake of microplastics into the human body, ranging from ng to mg per month. Accordingly, using 2.5 mg of particles for in vivo studies simulates an extreme exposure and certainly does not adequately reflect long-term, low-dosage microplastics intake to which most people in western countries are exposed. Additionally, the vast amount of microplastic particles in the environment is considered to be taken up by inhalation or ingestion. Thus, using an intravenous injection for administration as in our study indicates an artificial exposure. Yet, we decided for this experimental setup to provide first data on the effects of PS particles being present in the circulation, thereby directly interacting with leukocytes and endothelial cells. In addition, we are only using one type of well-defined microplastic particles (PS, 1 μm, carboxylated), whereas microplastics in our environment are very heterogeneous in terms of material, size, surface texture and charge. In the environment, microplastic particles absorb many other potentially toxic substances such as nitrogen oxides, bacterial products, toxic organic chemicals or heavy metals due to their surface properties [22]. Thus, their inflammatory effects are probably higher than that of the sterile particles used in this study. Further studies are needed to evaluate the effects of low-dosage and long-term administration of microplastic particles of different material using for instance an oral administration route in preclinical models, also in regard to vascular inflammation and disease.

Our data show enhanced inflammatory cytokine and adhesion molecule expression in vitro and in vivo. These findings are in line with other in vitro studies observing enhanced intestinal gene transcription of classical inflammatory pathways like nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) [17] or production of reactive oxygen species and elevated lipotoxicity in macrophages [12] after stimulation with polystyrene. Furthermore, in vivo we observed an increase in hepatic mRNA expression of the acute-phase proteins Saa1, Saa2 and Saa3 after polystyrene microplastic administration, representing a well-known reaction of the liver to inflammatory stimuli [16]. This observation is consistent with other studies showing hepatoxic effects of orally administered polystyrene in mice and rats [8, 23]. Of note, it was shown that photodegradation of polystyrene microplastic results in elevated hepatic inflammation and toxicity in the marine organism Epinephelus moara, suggesting that weather conditions such as solar radiation could even increase the hazard potential of polystyrene microplastic on aquatic organisms [24, 25].

In addition to already identified triggers of vascular inflammation including bacterial components during infection, reactive oxygen species or modified lipoproteins, this study suggests PS particles to be another, yet not described trigger initiating endothelial cytokine expression and leukocyte adhesion. Taking into account the observations mentioned above, potential aggravating effects of these particles in a scenario of ongoing inflammation, for instance present in cardiovascular patients, need to be addressed further. Of note, the identified interaction between polystyrene microplastic particles and neutrophils in the peripheral blood is indicative of an innate immune response, the consequences of which require further investigations.

In summary, for the first time we here show that PS particles are capable of activating the endothelium with subsequent monocyte adhesion. Taking into consideration that this process is not only important for the immune response, but also a hallmark of the initiation of atherosclerosis, microplastics need be evaluated critically as a novel environmental risk factor for cardiovascular disease and a general risk assessment is needed.

Raman spectrum and size measurement of PS particles.

(A) Raman spectrum of PS particles used in this study (B) Raman spectrum of PS particles as a reference, obtained at ramanlife.com. (C) Size distribution of PS particles were assessed by dynamic light scattering and presented as percent intensity.

(TIF)

Click here for additional data file.

Time- and dose-dependent cytokine expression of J774A.1 and MyEND cells after PS particle stimulation.

(A) Pilot study experiments (n = 2) in J774A.1 (top) and MyEND (bottom) cells using different time points (3 and 6 hours with a concentration of 107 particles/mL) and concentrations of PS particles (103, 105 and 107 particles/mL for 3 hours in J774.A1 cells and 6 hours in MyEND cells) to investigate kinetic and dosage effects. (B) Representative brightfield microscopy image showing a MyEND cell monolayer stimulated with PS particles at a concentration of 107 particles/mL. Scale bar = 20 μm.

(TIF)

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(XLSX)

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31 Aug 2021

PONE-D-21-23667

Polystyrene microplastic particles induce endothelial activation

PLOS ONE

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Reviewer #1: In this study the authors demonstrate that polystyrene microplastics induce endothelial activation through in vivo and in vitro mouse model experiments. Although the results are very interesting, the study is very poorly presented with no real discussion of the data. Therefore, I request major revision for this study.

Following several requests:

The abstract is really badly written and poorly organized. For example:

Line 30: In my opinion, it is improper to use “substance” referring to plastic items. Please, revise the sentence.

Line 31: “Small particles”. What do you mean? Please, insert the size.

Line 31-36: Rather than talking about the microplastics classification (put it in the intro), it would be more appropriate to explain the premise of this work in detail. In addition, it would be important for readers to adds details on the methods (Have you performed an in vitro study? In vivo study? Etc.)

Line 50 and 55: Please, refer to the document “Plastic Europe, 2020” not to the website.

Line 54-55: At least one citation is required!

Line 55: Please, check the English sentence!

Line 57: Maybe the citation [3] is inappropriate. Please, there are thousands of citation that you can use.

Line 65-67: …PS also led to the aggregation and endothelial adhesion of RBC (Please, see Barshtein et al., 2016 doi: 10.1007/s12013-015-0705-6).

Line 80-81: You have used in your study carboxylated MPs but you never mentioned before. Please, check: Murano et al., 2021 (doi: 10.3389/fmars.2021.647394). Use it for explaining the relevance of carboxy-modified particles in environment.

Line 86: Why was such a high concentration of particles used in this study? Please, add an explanation.

Line 211: Just curiosity. Did you find the particles only in the liver or in other organs as well?

The discussions need to be expanded. There is no part of comparison with other studies in the literature or general considerations also made on marine organisms for example.

Another suggestion is related to the title: why use the word “particles” if microplastic is a particle?

Reviewer #2: Dear Authors,

this is an interesting manuscript which for the first time highlights, through in vitro experiments and using mouse models, some new aspects of the deleterious effects of microplastics at the vascular level. In particular, the Authors found that polystyrene microplastic particles (PS) induce activation of endothelial cells (increased expression of adhesion molecules and leukocyte interaction) and increases the expression and release of inflammatory cytokines from monocytic cells. They also demonstrate PS uptake by circulating neutrophils.

Although the topic is very interesting, some points are not clear.

Here are my comments:

• MATERIALS AND METHODS: Line 73: The "MyEND" cells as reported in reference number 15 is a cell line: it should be specified. Line 81: TRICT is an acronym and I suggest specifying Tetramethylrhodamine the first time it is mentioned. Line 96: An important point that was not mentioned and that the Authors must specify is how many animals were used, this is important for the data statistical significance, especially for the experiments performed on blood, liver and aortic tissue samples. Line 109: which genes were evaluated, and which primers were used? It must be specified in the methods before finding them cited in the results and in the figures legends.

• RESULTS: Figures S2: The Authors performed only 2 experiments even though 3 experiments would have been better to reach statistical significance but defining it as a pilot study this could be accepted. However, in the legend it is not clear at 3 and 6 hours which PS concentration was used, specify as in the results 107. In J774A.1 cells, interleukin 1beta increases more at 3 hours than at 6 hours which is like the control; how do the authors justify this? Why did the authors choose 3 and 6 hours? It would have been interesting to evaluate longer stimulation times to visualize a chronic effect (like 24, 48, 72 hours of stimulation) which is what happens in real life. In fact, the chronicity of encountering microplastics leads to diseases. The authors have discussed this (Lines 263-266) but they could expand this concept by adding some references about it as well.

Figure 1: Does n = 7-10 refer to the number of experiments conducted?

Figure 2: Authors should show more representative images of the reported data (especially for B)

Figure 3A: It is low resolution, improve the resolution. Line 213 add a reference at the end of the sentence "indicating a hepatic inflammatory response to the PS particles".

Figure 4 and Line 229: ICAM-1 does not reach statistical significance, please specify that it is a trend.

Line 238: does n = 7 / group mean 7 mice per group? see comment on the number of mice in the comments on materials and methods.

Reviewer #3: In this study Ann-Kathrin Vlacil and collaborators demonstrate that microplastics are new environmental risk factors for endothelial inflammation.

The topic of this paper is very interesting and data convincing. Only few issues should be addressed in order to improve this study for the readers of PlosOne:

1. In Material and Methods, “Cell Culture and Treatment” paragraph, cells were exposed for 3 and 6 hours to PS particles (as also reported in suppl figure 2), instead in “Cell viability assay” and “adhesion assay” the exposure is for 16 hours. Please better clarify the exposure time to PS particles. What is the rationale in choosing 3, 6 and 16 hours? Probably a time course with the addition of longer exposure could be appropriate. Please provide data in case author have performed experiments of longer exposure.

2. In figure 2 images of the total well representative of a bigger field should be added to have a more consistent idea of cell adhesion.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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19 Oct 2021

We thank all reviewers for their valuable suggestions and corrections that helped us to improve the quality of our manuscript. All concerns/comments have been addressed properly. Please find below our detailed response and the corrections that have been made. All corrections are highlighted in red color in the revised version of our manuscript. Thank you very much for consideration.

Reviewer #1

Reviewer: The abstract is really badly written and poorly organized. For example:

Line 30: In my opinion, it is improper to use “substance” referring to plastic items. Please, revise the sentence.

Authors: We have replaced the term substance with resource (line 30) and have better organized the abstract according to the reviewer's suggestions.

Reviewer: Line 31: “Small particles”. What do you mean? Please, insert the size.

Authors: We now provide a general definition of microplastics (line 31-33) and defined the particles used in this study more precisely (1 µm, line 36). In addition, we have performed size measurements of the used particles by dynamic light scattering to confirm the manufacturer's information (S1C Fig, line 90-92, line 400-401).

Reviewer: Line 31-36: Rather than talking about the microplastics classification (put it in the intro), it would be more appropriate to explain the premise of this work in detail. In addition, it would be important for readers to adds details on the methods (Have you performed an in vitro study? In vivo study? Etc.)

Authors: As suggested, we have moved the classification of microplastics, etc. to the introduction, described the intent of our study (line 35-38), and described the methods used in more detail (line 37-38).

Reviewer: Line 50 and 55: Please, refer to the document “Plastic Europe, 2020” not to the website.

Authors: We now refer to the document (line 54/55 and 59, ref#2).

Reviewer: Line 54-55: At least one citation is required!

Authors: We have restructured this part of the introduction. The information on the size of the particles is covered by new ref#1 (line 48-52).

Reviewer: Line 55: Please, check the English sentence!

Authors: We have rewritten this sentence (line 59-60).

Reviewer: Line 57: Maybe the citation [3] is inappropriate. Please, there are thousands of citation that you can use.

Authors: The paper was replaced by Isaac MN et al. (line 61, new ref#5).

Reviewer: Line 65-67: …PS also led to the aggregation and endothelial adhesion of RBC (Please, see Barshtein et al., 2016 doi: 10.1007/s12013-015-0705-6).

Authors: Thank you for pointing this out, we have included the findings and the reference (line 70-72, ref#14).

Reviewer: Line 80-81: You have used in your study carboxylated MPs but you never mentioned before. Please, check: Murano et al., 2021 (doi: 10.3389/fmars.2021.647394). Use it for explaining the relevance of carboxy-modified particles in environment.

Authors: We agree with the reviewer that this is an important information and have now regularly indicated in the manuscript that we used carboxylated PS particles (e.g. line 36, 74 etc.). We have also cited the suggested literature (ref#21) and a review article on that topic by Andrady AL (ref#22) and discussed the relevance of surface modification and charge (line 274-278).

Reviewer: Line 86: Why was such a high concentration of particles used in this study? Please, add an explanation.

Authors: We decided for this concentration according to our pilot dose dependency and time response study in vitro (Suppl. Fig. 2), showing greatest effects at 107/mL. This concentration corresponds to 5.4 �g/mL. Respective information for all used PS particle concentrations (pilot studies, cell viability) were now added to the Materials and Methods section (line 94-95) to facilitate comparability with other studies. In view of this, we used a rather lower particle concentration in our study (see e.g. ref#17 Wu et al. 2020 using 12.5 and 50 mg/mL). As can be seen in S2BFig, each cell is in contact with only a few particles at this concentration, which we have already described in the results section (line 196-199).

Reviewer: Line 211: Just curiosity. Did you find the particles only in the liver or in other organs as well?

Authors: So far, we have only examined a handful of organs/tissues in this regard. We found particles in spleen, bone marrow and adipose tissue as well. A comprehensive analysis of further organs/tissues is planned for the future. We provide some exemplarily pictures for the reviewer only in the attached word file.

Reviewer: The discussions need to be expanded. There is no part of comparison with other studies in the literature or general considerations also made on marine organisms for example.

Authors: We now provide a more detailed comparison with other studies in the literature, see line 269-274 (ref#19 and ref#20), line 274-275(ref#8 and ref#21), line 299-302 (ref#17 and ref#12) or line 305-310 (ref#8, ref#23, ref#24 and ref#25).

Reviewer: Another suggestion is related to the title: why use the word “particles” if microplastic is a particle?

Authors: The term microplastic particles is frequently used in the field (more than 2000 hits in PubMed). Furthermore, we believe that this name best reflects the type of our stimulation: namely, with a variety of defined particles.

Reviewer #2

Reviewer: MATERIALS AND METHODS: Line 73: The "MyEND" cells as reported in reference number 15 is a cell line: it should be specified.

Authors: Thank you for pointing this out, we now have clearly indicated in material and methods that MyEND cells as well as J77A4.1 are cell lines. In addition, we provided the accession where more details about the cells can be found (line 79-81).

Reviewer: Line 81: TRICT is an acronym and I suggest specifying Tetramethylrhodamine the first time it is mentioned.

Authors: We have specified TRITC (line 87-88).

Reviewer: Line 96: An important point that was not mentioned and that the Authors must specify is how many animals were used, this is important for the data statistical significance, especially for the experiments performed on blood, liver and aortic tissue samples.

Authors: This is certainly an important information. Since we show the individual data points in the figures, we had omitted this information in the first version of the manuscript. However, it is often difficult to identify the exact number from the figures. We have now indicated the animal numbers used (line 107). Please note, the number of animals may vary between figures because we did not use all mice in all experiments or if we had to exclude data based on CT values in the PCR analyses, or similar.

Reviewer: Line 109: which genes were evaluated, and which primers were used? It must be specified in the methods before finding them cited in the results and in the figures legends.

Authors: We now specified the genes which were evaluated by real-time PCR and listed the sequences of the corresponding primers in materials and methods (line 125-136).

Reviewer: RESULTS: Figures S2: The Authors performed only 2 experiments even though 3 experiments would have been better to reach statistical significance but defining it as a pilot study this could be accepted. However, in the legend it is not clear at 3 and 6 hours which PS concentration was used, specify as in the results 107. In J774A.1 cells, interleukin 1beta increases more at 3 hours than at 6 hours which is like the control; how do the authors justify this? Why did the authors choose 3 and 6 hours? It would have been interesting to evaluate longer stimulation times to visualize a chronic effect (like 24, 48, 72 hours of stimulation) which is what happens in real life. In fact, the chronicity of encountering microplastics leads to diseases. The authors have discussed this (Lines 263-266) but they could expand this concept by adding some references about it as well.

Authors: To be more precise, we now specify the stimulation concentration (107 particles/mL) and time points (3 and 6 hours), see line 405-407 in figure legend and line 195-196 in the result section. In these pilot experiments, we examined two early time points and selected the time point where we saw the best effects (3 hours for J774A.1 and 6 hours for MyEND). We have focused on the initial onset of pro-inflammatory cytokine expression as a clear result of microplastic stimulation and wanted to exclude secondary effects potentially arising during long time exposure. Long time experiments with both cell types under our culture conditions are not feasible because cells begin to detach after more than 24 hours (independent of PS particles). However, we of course agree with the reviewer that organisms are exposed to microplastic in a chronic manner and added two recent studies investigating long-term exposure in a marine ecotoxicological model organism (see line 269-274 and ref#19 and ref#20).

Reviewer: Figure 1: Does n = 7-10 refer to the number of experiments conducted?

Authors: Yes, the number of independent experiments is given.

Reviewer: Figure 2: Authors should show more representative images of the reported data (especially for B)

Authors: We have chosen larger image details or selected other images (please see also reviewer #3). The adherent monocytes on the images now better reflect the differences found in the quantitate evaluation, namely that PS increases the adherence of monocytes about 3-fold.

Reviewer: Figure 3A: It is low resolution, improve the resolution.

Authors: We have increased both the resolution and the image size to better identify the PS particles in the tissue.

Reviewer: Line 213 add a reference at the end of the sentence "indicating a hepatic inflammatory response to the PS particles".

Authors: We already had a reference to the hepatic inflammatory response (Sack et al., ref#16), which we have now given here. In addition, we extended the discussion in this context (line 302-305).

Reviewer: Figure 4 and Line 229: ICAM-1 does not reach statistical significance, please specify that it is a trend.

Authors: To be more precise, we now state that Icam-1 shows a trend towards elevated expression (line 251-252).

Reviewer: Line 238: does n = 7 / group mean 7 mice per group? See comment on the number of mice in the comments on materials and methods.

Authors: Yes, the indicated numbers refer to the number of mice per group. We have addressed this issue in material and methods as suggested, see line 107 and in addition we now specify this in more detail in the figure legends (line 243 and 260).

Reviewer #3

Reviewer: In Material and Methods, “Cell Culture and Treatment” paragraph, cells were exposed for 3 and 6 hours to PS particles (as also reported in suppl figure 2), instead in “Cell viability assay” and “adhesion assay” the exposure is for 16 hours. Please better clarify the exposure time to PS particles. What is the rationale in choosing 3, 6 and 16 hours? Probably a time course with the addition of longer exposure could be appropriate. Please provide data in case author have performed experiments of longer exposure.

Authors: As stated above, we choose the indicated stimulation time points according to our pilot study, showing highest mRNA expression of inflammatory cytokines after 3 hours (J774A.1) and 6 hours (MyEND). We discuss this in the results section (see line 193-196). In the adhesion assay, we chose 16 hours to allow adequate expression of the adhesion molecules at the protein level (translation and transport to the membrane). In addition, we have already had good experience with this timing in previous studies (e.g. doi: 10.3390/cells10082146). For the cell toxicity study, we again chose the same time period to exclude cytotoxic effects of the PS particles on the endothelial cells within these 16 hours of stimulation for the adhesion assay. We have now indicated this in the results section (see line 192-193 and line 216-218). In this study, we focused exclusively on effects of PS particles at early time points and not on longer effects in which secondary effects of PS particles may also play a role. In addition, long time experiments with both cell types under our culture conditions are not feasible because – independent of PS particles – cells start to detach after more than 24 hours. However, longer exposure times in the environment are obviously relevant, so we now discuss two already existing studies on long-term effects of microplastics on marine organisms (see line 269-274 and ref#19 and ref#20 and see also reviewer #2).

Reviewer: In figure 2 images of the total well representative of a bigger field should be added to have a more consistent idea of cell adhesion.

Authors: We have enlarged the image sections shown in both cases. In B (flow experiments), however, only slightly, because we were limited by the equipment of the flow chamber and microscope in terms of resolution and detection of the cells. The images were taken at the end of the washing step under flow conditions. We now show the actual image size on which the evaluations were made (please see also reviewer #2).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

1 Nov 2021

PONE-D-21-23667R1Polystyrene microplastic particles induce endothelial activationPLOS ONE

Dear Dr. Grote,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: The authors have adequately addressed my previous comments and improved the quality of the manuscript. However, some small issues could be addressed.

-First of all, in my opinion, even define plastic as a "resource" is somehow incorrect. Therefore, the authors have to review the first sentence of the abstract. Plastic is a material/a product ....

-"In vivo" and "in vitro" must be must be written to Italic character

-Check References

I really appreciate the example images And I hope the authors will provide further insights on this topic for future publications.

Reviewer #2: The Authors have been addressed all comments. Hence, the manuscript has been improved and I have not further comment.

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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2 Nov 2021

Reviewer #1

We thank Reviewer#1 to further improve the quality of our manuscript. All corrections are highlighted in red color in the revised version of our manuscript.

Reviewer: First of all, in my opinion, even define plastic as a "resource" is somehow incorrect. Therefore, the authors have to review the first sentence of the abstract. Plastic is a material/a product....

Authors: That is true, mineral oil would be the raw material in this context. We have decided to use the term material (line 30).

Reviewer: "In vivo" and "in vitro" must be written to Italic character.

Authors: We have now italicized both terms throughout the manuscript.

Reviewer: Check References.

Authors: We have checked again the correct use of all references.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Nov 2021

Polystyrene microplastic particles induce endothelial activation

PONE-D-21-23667R2

Dear Dr. Grote,

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Reviewers' comments:

8 Nov 2021

PONE-D-21-23667R2

Polystyrene microplastic particles induce endothelial activation

Dear Dr. Grote:

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PLOS ONE