# Tire particles of different sizes induce a proinflammatory response of varying intensity in lung cells

Abderrahmane Bouredji, Bogdan Muresan-Paslaru, Riadh Lakhmi, Raphaël Passas, Jérémie Pourchez, Valérie Forest

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Abderrahmane Bouredji, Bogdan Muresan-Paslaru, Riadh Lakhmi, Raphaël Passas, Jérémie Pourchez, et al.. Tire particles of different sizes induce a proinflammatory response of varying intensity in lung cells. Journal of Environmental Sciences, 2025, 158, pp.586-597. 10.1016/j.jes.2025.03.011. emse-05093928

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Research Article

# Tire particles of different sizes induce a proinflammatory response of varying intensity in lung cells

Abderrahmane Bouredji<sup>a</sup>, Bogdan Muresan-Paslaru<sup>b</sup>, Riadh Lakhmi<sup>c</sup>, Raphaël Passas<sup>d</sup>, Jérémie Pourchez<sup>a</sup>, Valérie Forest<sup>a,*</sup>

ARTICLE INFO

Article history:
Received 12 September 2024
Revised 7 March 2025
Accepted 9 March 2025
Available online 10 March 2025

Keywords:
Air pollution
Traffic emissions
Non-exhaust emissions
Tire and Road Wear Particles (TRWP)
Health effects
Proinflammatory response

ABSTRACT

Pollution from road traffic contributes significantly to air pollution through pollutants from exhaust emissions (gases and particles) and non-exhaust emissions (tire wear particles, brake wear particles and the resuspension of road dust). This research examined the hazard of tire particles (TP) and in particular evaluated the effect of TP size on lung macrophages. TP were obtained by cryogenic grinding of a tire and subsequent sieving to obtain four groups of particles (TP70, TP30, TP15, TP5) of different sizes with average diameters of 107 µm, 55 µm, 22 µm, and 6 µm, respectively. A complete physicochemical characterization was performed to determine the size distribution, chemical composition and morphology of these particles. We then investigated the proinflammatory response, oxidative stress and cytotoxicity induced in RAW264.7 cells exposed to four different TP concentrations for 24 h. TP had no direct effect on cytotoxicity, nor did they increase reactive oxygen species (ROS) production in the cells. However, TP induced a significant and size-dependent proinflammatory effect, which was particularly pronounced with small particles. Moreover, this effect was concentration-dependent.

© 2025 The Research Center for Eco-Environmental Sciences, Chinese Academy of

Sciences. Published by Elsevier B.V.

This is an open access article under the CC BY license

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Introduction

With > 4.2 million premature deaths worldwide in 2019, air pollution is a major concern for human health

(Brunekreef and Holgate, 2002; WHO, 2022). Data suggest that there are around 3 million premature deaths due to air pollution in countries with high population growth (Tiotiu et al., 2020). In particular, exposure to particulate matter (PM) is associated with harmful effects on humans (Orru et al., 2011), as well as with a reduced life expectancy (de Oliveira et al., 2012; Dockery et al., 1993; Pope III et al., 1995). It is also estimated that air pollution is responsible for 3 % of deaths from

ARTICLE INFO

<footnote><sup>a</sup> Mines Saint-Etienne, Univ Jean Monnet, INSERM, U1059 Sainbiose, Centre CIS, F-42023 Saint-Etienne, France</footnote>

<footnote><sup>b</sup> Université Gustave Eiffel, AME-EASE, F-44344 Bouguenais, France</footnote>

<footnote><sup>c</sup> Mines Saint-Etienne, Univ Lyon, CNRS, UMR 5307 LGF, Centre SPIN, F-42023 Saint-Etienne, France</footnote>

<footnote><sup>d</sup> Université Grenoble Alpes, CNRS, Grenoble INP, LGP2, F-38000 Grenoble, France</footnote>

<footnote>* Corresponding author.
E-mail: vforest@emse.fr (V. Forest).</footnote>
cardiovascular complications in adults and 1 % in children (Cohen et al., 2005).

Road traffic pollution is a major contributor to air pollution (Evangeliou et al., 2020). These pollutants consist of elements originating from exhaust emissions (gases and particulates) as well as those non-exhaust emissions (tire wear particles, brake wear particles and the resuspension of road dust) (Thorpe and Harrison, 2008; Zhang et al., 2023). While more stringent regulations led to substantial reduction of pollutants from exhaust emissions, there are currently no regulations for non-exhaust particles (Baensch-Baltruschat et al., 2020; Li et al., 2023). Exhaust emissions mainly include, but is not limited to, a particulate phase of black carbon (BC) and an organic phase containing nitrate, sulphate and ammonium (Kostenidou et al., 2021). Ultrafine particles (smaller than 100 nm) from gasoline and diesel combustion pose an increased toxicological risk due to their ability to adsorb volatile organic compounds and metals on their surface (Forest, 2021; Tyler et al., 2016). In addition, polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion and simultaneously with the particulate phase, and their emission levels are comparable to those of exhaust gases, while they are more genotoxic and have carcinogenic effect (Muñoz et al., 2018). Studies have shown an association between increased mortality and overexposure to PM₁₀, as well as a higher risk of cardiovascular complications and mortality associated with overexposure to PM₂.₅ (Xing et al., 2016; Zanobetti et al., 2009). As a result, prolonged exposure to fine particulate matter in the air is likely to contribute to the development of serious diseases, including cardiovascular, lung diseases and cancer (Brunekreef and Holgate, 2002; Tainio et al., 2021; Valavanidis et al., 2008).

With the rising of a large fleet of electric vehicles, non-exhaust emissions are now steadily supplanting those from internal combustion engine vehicles, particularly in urban areas (OECD, 2020). Also, a recent study suggests that non-exhaust particle emissions may be higher in electric vehicles than in those equipped with internal combustion engines. This phenomenon is primarily attributed to the increased weight of vehicles due to their electric propulsion system, which significantly impacts the particle emission factor (Liu et al., 2021). Specifically, the contact of the tire with the road leads to the formation of tire and road wear particles (TRWP), which make up for a significant proportion of non-exhaust particulate emissions and have variable physical and morphological traits. Their diversity is the result of various factors, including their chemical composition, the type and nature of the tire tread which can vary from one tire to another, the road conditions, and the contaminants present on the road surface. Their emission level is also influenced by the driving style, the type of vehicle and its weight, as well as the chemical composition of the tire (Baensch-Baltruschat et al., 2020; Zhang et al., 2023). For example, it has been shown that the tire wear rate for hybrid vehicles was higher than for conventional vehicles, namely 72 and 53 mg/(veh·km) respectively (Liu et al., 2022a). In addition, unfavorable weather conditions, the environments surrounding roads and human influences contribute to the resuspension of elements on the road surface, which then enter into the composition of TRWP (Thorpe and Harrison, 2008). Further-

more, these particles make up a significant proportion of the primary and secondary microplastics distributed in the environment (Lassen et al., 2015; Magnusson et al., 2016) and their size varies from <0.1 µm, which allows them to penetrate the alveoli (Löndahl et al., 2006), to several tens or hundreds of microns. Moreover, De Oliveira et al. (2024a) analyzed sediments from a pond located near an urban ring road to investigate TRWP infiltration. The study reveals that a small portion of these particles disperses into the atmosphere, while the majority is transported to aquatic systems. Furthermore, the particles exhibit a bimodal distribution, with sizes ranging from 36 to 500 µm. Notably, the study indicates that larger particles found in the overflow zone may result from the aggregation of finer TRWP. Over time and through sedimentation, these particles can undergo mineral encrustation, promoting their aggregation. It is therefore crucial to assess the toxicity of TRWP emissions.

The impact of TRWP and brake wear particles (BWP) on human health has been previously examined in Bouredji et al. (2023) and Forest and Pourchez (2023). Analysis paid particular attention to understanding the mechanisms of toxicity which were mainly investigated using *in vivo* and *in vitro* models. It revealed the importance of factors such as the chemical composition, size distribution and generation mechanisms of emissions, which play a crucial role in modulating their effects in toxicological tests. Despite the discrepancies between the results of the different studies, the general conclusions point to a common mechanism of human toxicity through oxidative stress and proinflammatory response. Furthermore, the analysis has underlined the limitations of these studies while emphasizing the importance of working towards the goal of standardizing tests to better characterize the toxicity of TRWP (Bouredji et al., 2023). Finally, it was clearly emphasized that the assessment of TRWP toxicity in mammalian models is still limited and further comprehensive experiments are needed. To fill this gap, we propose an *in vitro* study investigating the toxicological profile of TP in a lung cell model. In particular, this work showed the influence of TP size on the induction of a proinflammatory response.

# 1. Materials and methods

## 1.1. Tire particles (TP)

The particles were produced by a cryogenic grinding process, with the tire supplied by the Gustave Eiffel University in France. It came from standard summer tires (Michelin™ Energy Saver, 185/60 R 15 88H). To ensure that the material tested was representative of the on-road tire tread condition, samples were taken from the treads of tires with a maximum wear rate of 40 % (>5 mm residual texture depth). In order to avoid overheating, the samples were slowly cut from three tires of the same brand using a ceramic blade. They were cleaned of surface incrustations using a damp cloth, ultrapure water and compressed air. They were subsequently stored at room temperature in dark conditions until the grinding step. The grinding procedure took place at the university Grenoble Alpes, LGP2, France, using the CryoMill shredder from Resch
(Verder International BV, Netherlands). The experimental pro-
tocol consisted of 10 grinding cycles performed in a zirconia
tank (25 mL) with 3 g of tire particles (≈1 mm). Each grinding
cycle lasted 2 min at a frequency of 30 Hz under liquid nitro-
gen, preceded and followed by a cooling phase. Subsequently,
a size-based separation by sieving was performed to obtain a
wide range of particle sizes. In this process, a 4-sieve tower
with separation sizes of 70, 30, 15 and 5 µm was used in con-
junction with a dedicated recovery tank. The sieving process
was carried out in 10-minute cycles at an amplitude of 2 mm
and an acceleration of 1 g, in the presence of polar solvents,
particularly ethanol.

Although the particles that enter the body via the respiratory tract are mainly particles smaller than 10 µm, they undergo physicochemical changes once they are in the organism. For example, the formation of a protein corona is well documented and aggregation/agglomeration phenomena can also occur. For this reason, cells can be confronted with particles larger than 10 µm. To reflect this size diversity, we examined a range of particle sizes and analyzed the response of cells to particles of the different sieved size fractions (6 to 107 µm).

1.2. Physicochemical characterization

1.2.1. *Morphological analysis by Scanning Electron Microscopy (SEM)*

The SEM images were taken with a Zeiss ULTRA-Plus micro-
scope equipped with a Field Emission Gun (FEG) and an in-
lens SE detector (Supra 55VP, Zeiss, France). The TP were de-
posited on a carbon ribbon and then metallized with gold (50 s
at 20 mA) for observation. All images were taken with sec-
ondary electrons collected by an in-lens detector at 20 kV and
a working distance of 10 mm.

1.2.2. *Elemental composition by energy-dispersive X-Ray spectroscopy (EDS)*

The elemental composition of the samples was determined
using EDS. The SEM used in this study was coupled with an
Ultim® Extreme Windowless 100 mm² silicon drift detector
(SDD) from Oxford Instruments (Xmax80, Oxford Instruments,
France). The elements were excited with a beam voltage of
20 keV and the identification was performed with the Aztec
software.

1.2.3. *Chemical analysis by inductively coupled plasma mass spectrometry (ICP-MS)*

Chemical analysis was performed using an Agilent 7800 In-
ductively Coupled Plasma Mass Spectroscopy (Agilent 7800
ICP-MS, Agilent Technologies, US) to quantify aluminum, cal-
cium, copper, cadmium, iron, sodium, lead, sulfur, antimony,
silicon, titanium, zinc and zirconium using internal standards.
The mineralization procedure began by carefully suspending
150 mg of powder in a mixture of 3 mL hydrochloric acid (HCl,
2 mol/L) and 1 mL nitric acid (HNO3). This suspension was
heated on a hotplate at 100 °C until it evaporated. Then, 3 mL
hydrofluoric acid (HF) was added, the Teflon-Savillex vessel
was hermetically sealed and the mixture was allowed to stand
at 120 °C for 48 h and then evaporated at 110 °C. Next, 2 mL of
HNO3 was added and evaporated at 110 °C. This step was re-
peated twice. Then, 2 mL HCl was added and evaporated at

110 °C. In the last step, 25 mL HCl was added and the final
weight of the beaker was measured.

1.2.4. *Particle size distribution*

The size distribution of TP was accurately characterized using
a laser diffraction size analyzer (Mastersizer 3000, Malvern In-
struments Ltd., Worcestershire, UK). The Hydro MV wet dis-
persion system with ethanol as the solvent was used for this
purpose. In addition, a 2-min sonication step was performed
prior to the analysis procedure.

1.2.5. *Specific surface area by Brunauer Emmett Teller (BET) measurement*

The specific surface area was determined by analyzing the ad-
sorption of krypton on a TriStar 3000 analyzer (Micromeritics,
US). The pulverized samples (0.88 g) were placed in the sample
holder and vacuumed for at least 24 h to ensure mass stability.

1.3. *Cell culture*

The RAW 264.7 cell line derived from mouse peritoneal macrophages was transformed by the Abelson murine leukemia virus and was provided by ATCC Cell Biology Collection (Promochem, LGC, Molsheim, France). We used lung macrophages as these cells of the immune system are the first line of defense of an organism against inhaled particles. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % of fetal calf serum and 1 % of penicillin-streptomycin at 37 °C under a 5 % carbon dioxide humidified atmosphere.

For TP/cell contact, cells were seeded in 96-well plates (100,000 cells in 50 µL of medium per well) and were allowed to adhere overnight. Particles were diluted in cell culture medium to reach the following final concentrations: 15, 30, 60 and 120 µg/mL. This concentration range was based on literature data (Drasler et al., 2017). Particles were added to the cells and further incubated for 24 h.

1.4. *In vitro toxicity assays*

1.4.1. *Cytotoxicity - Lactate dehydrogenase (LDH) release*

To evaluate cell membrane integrity, the release of cytoplas-
mic lactate dehydrogenase into the supernatant was assessed
using the CytoTox-96™ Homogeneous Membrane Integrity
Assay (Promega, Charbonnières-les-Bains, France) according
to the manufacturer's instructions. The optical density of the
samples was determined using a microplate reader (Multi-
skan RC; Thermo Labsystems, Helsinki, Finland) set to 450 nm.
Three independent experiments were performed, each in qua-
druplicate, and the activity of the released LDH was compared
to that of negative control cells (incubated without particles).
A positive control consisted of the maximal cellular LDH re-
leased after cell lysis.

1.4.2. *Proinflammatory response - TNF-α production*

After incubation with TP, the production of TNF-α was as-
sessed in the supernatant using a commercial ELISA kit
(Quantikine® Mouse TNF-α Immunoassay; R&D Systems, Lille,
France) according to the manufacturer's instructions. The op-
tical density of each sample was determined using a mi-
croplate reader (Multiskan RC; Thermo Labsystems, Helsinki,
Finland) set to 450 nm. A standard curve was established and results were expressed in picograms of TNF-α per milliliter of supernatant. Three independent experiments were performed, each in quadruplicate, and the production of TNF-α was compared to that of control cells (incubated without TP).

### 1.4.3. Oxidative stress - reactive oxygen species (ROS) production

A wide array of ROS activity can be assessed with the OxiSelect™ ROS Assay Kit (Euromedex, Mundolsheim, France). The assay uses the conversion of a non-fluorescent substrate, 2.7'-dichlorodihydrofluorescein diacetate that can easily diffuse through cell membranes and be converted into a fluorogenic molecule, 2.7'-dichlorodihydrofluorescein (DCF), in the presence of ROS, fluorescence amount is directly related to ROS levels. Fluorescence was detected using a Fluoroskan Ascent fluorometer (Ex: 480 nm, Em: 530 nm, Thermo Scientific™, US) after a 90 min and a 24 h incubation of cells with the particles. A positive control was included by incubating cells with H₂O₂ (1 mmol/L). Three independent experiments were performed, each in quadruplicate, and the generation of ROS was compared to that of the negative control (cells incubated without TP).

## 1.5. Statistical analysis

Analyses and graphics were conducted using GraphPad Prism® (version 9, GraphPad Software, San Diego, CA). Data are presented as mean ± standard deviation (SD). Statistical significance was assessed using two-way ANOVA tests. Differences were considered to be statistically significant when the *p*-value was <0.05, with significance thresholds set at: \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. Each data point reflects the mean from a minimum of three independent experiments, presented with the SD.

# 2. Results

## 2.1. Description of the TP samples

As can be seen in Fig. 1, we cryogenically ground the tire to obtain a TP powder (from now on referred to as TP total), followed by sieving to obtain TP fractions of different sizes for subsequent toxicological analysis. The TP fractions generated are hereafter referred to as TP, followed by their representative diameters in microns. We used four sieves with retention limits of 70, 30, 15, and 5 µm, respectively. In addition, a reservoir was installed to collect the particles falling through the last sieve. The TP70 group, comprising the largest particles, was collected using the 70 µm sieve, while the medium particles, referred to as TP30, were collected using the 30 µm sieve. The TP15 group is the sum of the particles recovered on the 15 and the 5 µm sieves. Finally, the TP5 group comprised the finest particles collected in the reservoir below the 5 µm sieve. It is important to note that TRWP collected under actual driving conditions often exhibit bi- or tri-modal particle size distributions centered at < 0.03 µm and 0.05-0.30 µm by number and <0.4 µm and 0.5-2 µm and >10 µm by mass (Beji et al., 2021;

<table><caption>Table 1 - Distribution of total size by volume density.</caption><thead><tr><td>Sample</td><td>Average size (µm)</td><td>D10* (µm)</td><td>D90** (µm)</td></tr></thead><tbody><tr><td>TP70</td><td>107</td><td>63</td><td>167</td></tr><tr><td>TP30</td><td>55</td><td>32</td><td>87</td></tr><tr><td>TP15</td><td>22</td><td>10</td><td>37</td></tr><tr><td>TP5</td><td>6</td><td>2</td><td>15</td></tr></tbody></table>

De Oliveira et al., 2024b). Thus, on a mass basis, the TP generated in this research primarily represent the coarse fraction of tire wear PM emissions to which populations and ecosystems near roads are exposed. Fig. 1 also shows the mass distribution of the individual TP size fractions within the total powder. TP70 made up the largest proportion of the composition with a share of 66 %, followed by TP30 with 23 %, TP15 with 7 % and TP5 with 4 %.

## 2.2. Physicochemical characterization of the TP

### 2.2.1. Particle size distribution

The particle size distribution was first evaluated in the total TP powder (in terms of volume and number, Fig. 2). The particle volume density distribution showed an average volume-weighted particle size of 113 µm, with a median particle size distribution (D50) of 108 µm. In addition, the analysis of the particle number distribution showed a median (D50) of 9 µm. This result emphasizes the significant presence of fine particles smaller than 10 µm, indicating that the sample covers a wide size range, from the largest to the finest particles. It is important to note that the BET analysis results indicate that the total powder has a relatively low specific surface area of 0.172 m²/g. This finding is consistent with SEM observations, which show an absence of apparent porosity in the particles.

The analysis of the particle size distribution within the TP70, TP30, TP15 and TP5 fractions is shown in Fig. 3. It is noteworthy that the average number distributions, for each group of particles are as follows: 17 µm for TP70, 11 µm for TP30, 0.8 µm for TP15, and 0.7 µm for TP5. The number-weighted average diameters of TP15 and TP5 fractions are therefore close to that of the submicron TRWP collected under actual driving conditions (i.e., 0.05-0.30 µm, see Section 2.1). These data emphasize the diverse size distributions achieved by the sieve separation technique and highlight its effectiveness in segregating particle samples of different sizes. The powder therefore consists primarily of coarse particles, but also includes a range of fine particles.

In addition, Table 1 shows the overall size distribution of the different samples. It presents the average size for each sample, as well as the size distribution indicators D10 and D90. These results reflect the efficiency of the separation and show that our particles do not exceed a maximum size of 167 µm.

### 2.2.2. Particle morphology

The morphology of the TP was analyzed before (TP total powder) and after size fractionation (TP70, TP30, TP15 and TP5)

<footnote>\* D10: means that 10 % of the sample is smaller than or equal to the D10 value.</footnote>

<footnote>\*\* D90: means that 90 % of the sample is smaller than or equal to the D90 value.</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1 – Illustrative diagram for obtaining the different tire particles (TP) size fractions. Created with BioRender.com.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2 – Particle size distribution by volume and number in total TP powder.</figcaption></figure>

using SEM (Fig. 4). The analysis clearly showed that the TP to-
tal powder exhibited particles with a remarkable variability
in size and geometrical shape caused by the cryogenic grind-
ing process (Fig. 4a). In addition, the analysis showed parti-
cle aggregation due to a pronounced electrostatic effect of the
fine particles deposited on the surface of the larger particles
(Fig. 4b). TP70 exhibited a certain degree of homogeneity, to-
gether with the presence of some small particles adsorbed on
its surface (Fig. 4c). Furthermore, the particles collected on the
15 and 5 µm sieves indicate that it is a dense sample charac-
terized by an abundance of small particles interspersed with
larger particles. In addition, it is noted that fine particles tend
to adhere to larger particles, potentially making it difficult to
delineate particle contours and resulting in a saturated ap-
pearance of the images (Fig. 4d).

2.2.3. *Chemical analysis*

The chemical composition of the TP was then analyzed on the
basis of TP total, initially using EDS spectroscopy (Fig. 5). The
results showed that TP total has a well-defined profile. A no-
table silicon peak emerged, highlighting its predominant pres-
ence in the tire composition (silicon is used as a filler). In ad-
dition, within the range of elements, sulfur (S) and zinc (Zn)
peaks emerged as distinctive markers characterizing TP.

The samples were also analyzed using ICP-MS. The re-
sults confirmed the elemental composition determined by the
EDS analysis. In particular, the considerable amount of sulfur,
which is mainly used in the vulcanization process, and zinc,
which serves as a catalyst in the treatment of tires, deserve
attention. Other elements such as iron (Fe), sodium (Na), cal-
cium (Ca) and aluminum (Al) were also detected.
<figure><img src="image_4.png" /><figcaption>Fig. 3 – Particle size distribution in TP70, TP30, TP15 and TP5 size-fractions. Analysis performed by dynamic light scattering (DLS) based on volume density (a) and number density (b).</figcaption></figure>

## 2.3. Toxicological analyses

### 2.3.1. Cytotoxicity

After exposing the cells to TP concentrations of 15, 30, 60 and 120 µg/mL for 24 h, cytotoxicity was assessed by LDH released into the culture medium. As Fig. 6 shows, incubation of the cells with the TP total powder or the different size fractions of TP did not lead to a significantly increased LDH release compared to the control cells (unexposed). This indicates that TP did not induce cytotoxicity.

### 2.3.2. Oxidative stress analysis

Fig. 7 shows the relative amount of ROS produced by cells exposed to the whole TP powder or the different

TP size fractions. The results clearly show that TP, regardless of their size, did not induce ROS production that was statistically different from that observed in control cells, neither after 90 min nor after 24 h of incubation. These data suggest that TP did not induce oxidative stress.

### 2.3.3. Proinflammatory response

Fig. 8 shows the relative TNF-α production observed after incubation of cells with TP. While TP total powder, TP5 and TP70 did not significantly increase TNF-α production compared to control cells, TP15 induced a significantly increased TNF-α production, which was dose-dependent. Similarly, cell incubation with TP30 led to a dose-dependent increase in
<figure><img src="image_5.png" /><figcaption>Fig. 4 – Observation of particles from the TP total powder at X70 magnification (a) and X558 magnification (b), particles from TP70 observed at X488 magnification (c) and from TP15 shown at X1680 magnification (d).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5 – EDS elemental analysis of TP revealing the distinctive peaks of the trace elements identified, obtained at an excitation voltage of 20 keV. CPS: counts per second per electron volt. The presence of gold (Au) is due to the metallization step.</figcaption></figure>

TNF-α production, although this was only statistically sig-
nificant at the highest dose (120 µg/mL). These data sug-
gest a TP size-dependent induction of a proinflammatory re-
sponse, although further studies are needed to confirm this
effect (assessment of other cytokines and the antioxidant
system).

3. Discussion

The present study has shown that generated TP do not induce
significant cytotoxicity on lung cells nor increased ROS pro-
duction, while they trigger a size-dependent proinflammatory
<figure><img src="image_7.png" /><figcaption>Fig. 6 – Evaluation of cytotoxicity of the TP total powder and the different TP size-fractions - analysis after 24 h of particle exposure. Relative lactate dehydrogenase (LDH) release calculated as the ratio of LDH released from treated cells to LDH released by control (unexposed) cells. Results are means of three independent experiments. C-: Negative control. C+: Positive control. Statistical significance (*** P < 0.001) was determined by two-way ANOVA inter-group comparisons.</figcaption></figure>

response. These observations are partially consistent with the literature. Indeed, some studies have shown that TP were able to induce cytotoxicity. In particular, Poma et al. (2019, 2023) and Jiang et al. (2023) reported a significant induction of apoptosis in alveolar macrophages and human airways organoids after exposure to TP.

ROS production remains controversial as we demonstrated that generated TP do not increase cellular ROS production, like Karlsson et al. (2006) who exposed human lung epithelial cells (A549) to TP, but in contrast to Lindbom et al. (2007) and Jiang et al. (2023) who showed the ability of TP to induce oxidative stress in macrophages and human airways organoids.

Consistent with our findings, the capacity of TP to induce a proinflammatory effect seems commonly acknowledged and it was clearly evidenced by the release of cytokines in macrophages (Gustafsson et al., 2008; Karlsson et al., 2006, 2011; Lindbom et al., 2006, 2007; Poma et al., 2019), lung epithelial cells (Karlsson et al., 2006; Lindbom et al., 2006) and human airways organoids (Jiang et al., 2023). Moreover, the proinflammatory effect of TP has also been demonstrated in vivo, when mice or rats were exposed to TP (Gottipolu et al., 2008; Li et al., 2023; Mantecca et al., 2009, 2010).

The observed discrepancies in cytotoxicity and oxidative stress can be partly explained by different parameters. First of all, the type of samples should be taken into account. Indeed, different methods of particle collection are used in the literature (Bouredji et al., 2023). Some studies rely on direct sampling of particles on the road, while others use road simulators. In addition, some studies obtain particles by direct grinding of tires. Depending on how tire particles are produced, their composition and potential toxicological profile differ. TRWP consist of tire segments and road material originating from the contact zone as well as external contaminants such as resuspended pollutants. TP produced by grind-

ing yields micrometric fragments in considerable quantities, whereby the grinding process preserves the chemical integrity of the original tire material. The extraction of TP using road simulators allows precise control of the parameters of speed, abrasion and pressure, so that the particles are produced in smaller quantities but are more representative of real-life conditions. This method ensures that the particles produced are not contaminated by external pollutants. Finally, the study of TRWP that originate directly from the environment is another approach that allows an in-depth analysis of their impact on human health, as they more fully reflect real-world emissions. In this context, Kreider et al. conducted a comparative study on the morphology and chemical composition of TRWP with particles generated in a laboratory traffic simulator and with particles from the tread of floor tires. The preliminary results of this research emphasized the morphological and compositional subtleties of TRWP. TRWP were found to contain elevated concentrations of substances associated with road and environmental pollutants due to their synthesis process (Kreider et al., 2010).

Similarly, Poma et al. (2019) highlighted the influence of the nature of the samples on their toxicity, focusing on the comparison between particles originating from car or truck tires. These particles elicited a significant, dose-dependent proinflammatory response that was more intense in TP from cars than in TP from truck, with the latter eliciting an earlier but weaker effect. The analysis suggests that the composition of TP, defined by the ratio of natural rubber to synthetic polymers, influences its toxicity. The presence of natural rubber, whose main component is cis-polyisoprene, in large quantities in lorry particles has a less strong effect compared to synthetic polymers (such as butadiene), which are more common in car tires (Poma et al., 2019). Consequently, the nature and type of tire composing the particles can influence interactions
<figure><img src="image_8.png" /><figcaption>Fig. 7 – Analysis of relative reactive oxygen species (ROS) production in response to different TP sizes at 90 min (a) and 24 h (b). Results are expressed as the ratio of ROS produced by treated cells to ROS released by control cells, and represent the means of three independent experiments. C-: negative control. C+: positive control. Statistical significance (*** P < 0.001) was determined by two-way ANOVA inter-group comparisons.</figcaption></figure>

with the organism and induce a significant proinflammatory response.

In addition, TP released into the environment may undergo changes such as ageing, leaching, etc. and their toxicological profile may change accordingly. In this context, Liu et al. (2022b) have shown that photoaged TP incubated with alveolar macrophages caused a decrease in cell viability, increased oxidative stress and increased secretion of proinflammatory factors. Similarly, several studies have investigated eluates, organic extracts or even elastomeric derivatives of TP. Gualtieri et al. (2005a) analyzed eluates and organic derivatives of TP and found significant cytotoxicity at high concentrations as well as a dose-dependent decrease in cell proliferation. They also showed that TP extracts curtailed both

cell growth and viability within the A549 cell lineage in a dose-responsive manner, manifesting a lethal outcome post 48 h (Gualtieri et al., 2005b). Moreover, pursuing their inquiries into TP extracts, Gualtieri et al. demonstrated that these extracts inflicted cellular damages akin to those attributed to PM₁₀ air pollutants, primarily through the escalation of ROS within the cells (Gualtieri et al., 2008). In addition, other studies analyzing organic extracts of TP debris in A549 cells have shown that exposure to these TP enhances necrotic cytotoxicity and leads to an increase in LDH levels in the extracellular environment. TP also led to a weakening and alteration of cell membranes through changes in lipid microdomains (Beretta et al., 2007).

Our results emphasize the influence of TP size on the inflammatory response. Tire particles of medium size, TP15 (av-
<figure><img src="image_9.png" /><figcaption>Fig. 8 – Analysis of the proinflammatory response induced by different TP size-fractions as assessed by TNF-α levels measured after 24 h of exposure. Results are expressed as the ratio of TNF-α produced by treated cells to that produced by control cells, representing means of three independent experiments. Statistical significance (* P < 0.05; ** P < 0.01; *** P < 0.001) was determined by two-way ANOVA inter-group comparisons. C-: negative control.</figcaption></figure>

erage size 22 µm), show a significantly increased proinflam-
matory effect, which escalates in a dose-dependent manner.
Conversely, the larger TP30 particles (average size 58 µm) only
show a significant effect at higher concentrations. However,
this specific inflammatory response was not observed for the
particles at the ends of the size distribution, namely TP70 and
TP5. The analyses suggest that the inflammatory potential of
TP is closely related to their composition. Furthermore, the
fact that only two particle sizes cause an inflammatory reac-
tion, while other sizes do not have the same effect, underlines
the importance of further analyses on the influence of parti-
cle size. However, such results were not evident for TP total,
which contains both small and large particles. This discrep-
ancy could be explained by the proportions of the different
fractions within the powder, especially since the largest par-
ticles (TP70) are the most prevalent in the sample. The lack of
a direct effect with larger particles, such as TP70, can be at-
tributed to the fact that they are larger than the macrophages
and therefore preclude direct interaction. On the other hand,
the minimal effect observed with TP5 particles could be due to
their high electrostatic potential and robust aggregation abil-
ity, possibly leading to the formation of larger particle con-
glomerates that could prevent direct cellular interactions.

**4. Conclusions**

In the present study, we were able to demonstrate that
TP triggered a size-dependent proinflammatory response in
macrophages. However, TRWP are more complex than TP, and
further research is needed to study these samples more rep-
resentative of real emissions. Indeed, TRWP are increasingly

recognized as significant pollutants among non-exhaust PM
emissions. However, the complicated nature of TRWP makes
both the accurate measurement of their emissions and the as-
sessment of their health effects difficult. This complexity is
exacerbated by the simultaneous emission of other pollutants
from road transport, which significantly affects the analysis of
health risks (Forest, 2021; Fussell et al., 2022; Piscitello et al.,
2021). Furthermore, the task of distinguishing the effects of
direct emissions from those of dispersed particulate mat-
ter is a major challenge. This distinction is crucial to under-
stand the dynamics of additive, synergistic or antagonistic ef-
fects, which complicates the accurate assessment of potential
health risks (Piscitello et al., 2021).

**Declaration of competing interest**

The authors declare the following financial interests/personal
relationships which may be considered as potential compet-
ing interests:

Abderrahmane Bouredji reports financial support was pro-
vided by The French Agency for Ecological Transition. Bogdan
Muresan reports a relationship with Michelin that includes:
funding grants. Jeremie Pourchez reports a relationship with
Michelin that includes: funding grants. Valerie Forest reports
a relationship with Michelin that includes: funding grants. If
there are other authors, they declare that they have no known
competing financial interests or personal relationships that
could have appeared to influence the work reported in this
paper.
CRediT authorship contribution statement

**Abderrahmane Bouredji:** Writing – original draft, Visualization, Investigation, Formal analysis. **Bogdan Muresan-Paslaru:** Writing – review & editing, Resources, Investigation. **Riadh Lakhmi:** Writing – review & editing, Validation, Supervision, Resources, Formal analysis. **Raphaël Passas:** Writing – review & editing, Resources, Methodology. **Jérémie Pourchez:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Conceptualization. **Valérie Forest:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization.

Acknowledgments

This work was supported by the Ecole des Mines Saint-Etienne and the Agence de l'Environnement et de la Maîtrise de l'Energie.

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