Exposure to diesel particulates induces an immunosuppressive microenvironment that promotes the progression of lung cancer.

Introduction
Air pollution is a major environmental and public health concern. Particulate matter (PM), classified by aerodynamic size, is a major airborne pollutant that penetrates deep into the lungs and is linked to multiple adverse effects, such as cardiovascular diseases, strokes and lung cancer [1]. Epidemiological studies conducted on large cohorts have established a correlation between exposure to inhaled fine particles measuring ≤2.5 µm (PM2.5) and all-cause mortality [2]. Diesel exhaust particles (DEP) are a key component of PM2.5 in air pollution, and they have been classified as class 1 carcinogens to humans by the International Agency for Research on Cancer (IARC) [3]. However, beyond these well-established mechanisms, the influence of DEP on the pulmonary immune microenvironment, and its potential interference with anti-tumour immune responses remains poorly understood.
Polymorphonuclear neutrophils (PMNs) identified in mice as Ly6G+Ly6C+CD11b+ cells represent the most abundant population of innate immune cells in circulation. They play a central role in the early response to pathogens through phagocytosis, degranulation and NET formation. In addition to their antimicrobial functions, PMNs also contribute to immune regulation by producing cytokines and chemokines and engaging in crosstalk with other immune cell populations [4]. DEP exposure induces the recruitment of PMNs to the lung, where they play a key role in the pathogenesis of air pollution-related diseases, notably by promoting the formation of neutrophil extracellular traps (NETs) [5]. Whereas PMNs are primarily involved in host defence against pathogens and tissue remodelling, their regulatory role on adaptive immunity has received growing attention in recent years. PMNs play a dual role in cancer: while some subsets contribute to anti-tumour immunity, others can promote tumour progression and suppress T cell responses [6]. The immunosuppressive activity of PMNs has been largely attributed to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), a subset of pathologically activated PMNs that accumulate in cancer. These cells have been shown to dampen immune responses and contribute to the formation of an immunosuppressive microenvironment, which may facilitate immune evasion and tumour progression [7]. Transcriptomic analyses have identified CD14, recognized as a marker of monocytic cells, as a signature associated with the immunosuppressive potential of PMNs in mice, distinguishing specific neutrophil subsets. The expansion of these CD14pos PMN-MDSCs plays a key role in immunosuppression by limiting T cell proliferation [8]. This inhibition drives T lymphocytes into a dysfunctional state in which they lose their ability to mount effective immune responses. We hypothesized that DEP exposure may contribute to an immunosuppressive environment that promotes lung tumour progression. To address this hypothesis, we employed acute DEP exposure in wild-type (wt) mice and chronic DEP exposure in KrasLSL-G12D/+-Trp53lox/lox mice (KP mice), in which we also evaluated changes in spontaneous tumour development. We characterized the heterogeneity of PMNs recruited in the lung after acute and chronic DEP exposure and assessed their immunosuppressive phenotype using flow cytometry and functional assays. In parallel, we analysed the phenotype of infiltrating T cells to evaluate the broader impact of these changes on the tumour immune microenvironment. Given the rising global burden of air pollution and lung cancer, elucidating how DEP exposure alters the immune landscape is crucial for uncovering mechanisms that drive tumour progression and for identifying therapeutic strategies. These may include limiting the recruitment of immunosuppressive neutrophils, adapting treatments to PM-induced immune alterations, or protecting patients from further pollutant exposure during therapy.

Materials and methods

Mice
Animal procedures were conducted in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) and all experiments had previously been approved by the Animal Ethical Committee of the University of Liege (protocol references: # 2136). C57BL/6 mice (male and female, 6–8-week-old) were purchased from Janvier’s Labs (France). KrasLSL-G12D/+-Trp53lox/lox (KP) mice were obtained from Dr Pierre Close’s laboratory at University of Liège (male and female mice aged 12-16 weeks were used). All mice were housed and bred in institutional specific pathogen-free facilities at the GIGA Institute (Liège, Belgium), maintained in a 12-hour light-dark cycle and had access to normal chow diet and water ad libitum.

Intratracheal instillation of DEP into mouse lungs
Details are provided in the online supplementary material and methods section.

Lung cell and spleen cell isolation
Details are provided in the online supplementary material and methods section.

T cell isolation from lymphoid tissues
Lymphocytes were isolated by mechanically disrupting lymph nodes from naïve C57BL/6 mice using a flat-bottomed syringe, followed by collection in PBS-EDTA. Pan T cells were then isolated using the Pan T Cell Isolation Kit (Miltenyi Biotec) in accordance with the manufacturer's instructions, and separation was performed using LS columns on a QuadroMACS separator (Miltenyi Biotec).

Flow cytometry staining
Cells were first incubated for 15 min with Fc Block (anti-mouse CD16/32, clone 93, BioLegend) diluted in FACS buffer (PBS with 0,5 % BSA). Surface marker antibodies were resuspended in FACS buffer (PBS with 0,5 % BSA) and cells were incubated for 30 min with the corresponding fluorochrome-conjugated antibodies (listed in the key resources table). Cells were washed with PBS and stained with fix viability staining Zombie Aqua, Violet, or NIR (BioLegend) resuspended in PBS and incubated for 20 min. Cells were then washed with PBS and resuspended on FACS buffer or follow intracellular staining of target proteins.
For intracellular staining of cytoplasmic proteins, cells were fixed and permeabilized using Cyto-Fast Fix/Perm buffer (BioLegend) according to the manufacturer’s instructions. Antibodies were diluted in perm buffer and incubated with the cells for 30 minutes.
For intranuclear staining of proteins, cells were fixed and permeabilized using Foxp3/transcription factor staining kit (eBioscience) according to the manufacturer’s instructions. Antibodies were diluted in the permeabilization buffer of the kit and incubated with the cells for an hour.
Flow cytometry acquisition was performed on BD LSR Fortessa using FACSDiva software, and the data was processed with Flow Jo software. Gating strategies to identify the different cell populations are provided in Figure S1-S3.

Isolation of CD14neg and CD14pos PMNs
Mouse neutrophils (PMNs) were isolated from lung single-cell suspension prepared as previously described. The cell suspension was incubated with Anti-Ly-6 G Microbeads (Miltenyi Biotec) and passed through MACS LS columns (Miltenyi Biotec) on a QuadroMACS separator (Miltenyi Biotec) to capture neutrophils, following the manufacturer's protocol. Neutrophils were eluted by gently plunging the LS columns and subsequently stained with anti-Ly6G, anti-CD14 and the viability marker SYTOXTM blue. They were then sorted using a BD FACS Aria (BD Biosciences) as CD14 negative or positive neutrophils. The isolated PMNs were then used for further analysis.

Cytospin of neutrophils
After FACS sorting of CD14neg and CD14pos PMNs, cytospins were prepared using a StatSpin CytoFuge 2 (IRIS compagny). In detail, 2.5 × 104 cells were centrifuged (4400 rpm, 2 min) onto Superfrost microscope slides (Epredia) and dried overnight at room temperature. Cells were then fixed in methanol and stained with hematoxylin and eosin (H&E). Image documentation was performed using the NanoZoomer 2.0-HT slide scanner system (Hamamatsu) and the NanoZoomer Digital Pathology software.

T cell suppression assay
CD14neg and CD14pos PMNs were FACS sorted as described above. T cells were isolated as previously described, then washed in PBS and labelled with CellTrace Violet (Invitrogen) following the manufacturer’s instructions. To activate the T cells in culture, a 96-well tissue culture plate was pre-coated with anti-mouse CD3 antibody (5 µg/ml; eBioscience) overnight at 4°C, then washed with PBS. Neutrophils and T cells were co-cultured at a 1:1 ratio or alone as controls in 200 µl RPMI medium (Lonza) per well, supplemented with 10 % FBS (Gibco), 50 U/ml penicillin-streptomycin (Gibco), 1 % MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and 0.05 mM 2-mercaptoethanol (Gibco). Anti-mouse CD28 (1 µg/ml; eBioscience) was also added to the culture to provide co-stimulatory signalling on T cells. The cells were incubated at 37°C in a 5 % CO2 environment for 72 hours, after which T cells were collected from the supernatant, stained for surface markers (anti CD3 and CD8) and viability. Finally, proliferation of CD8+
T cells was assessed using a BD LSR Fortessa flow cytometer.

Quantitative real time polymerase chain reaction (qRT-PCR)
Details are provided in the online supplementary material and methods section.

Identification of NETs In Vivo and In Vitro
Details are provided in the online supplementary material and methods section.

Collection of bronchoalveolar lavage fluid (BALF)
After sacrifice, a bronchoalveolar lavage was performed by injecting 3 × 1 ml of PBS-EDTA 0,05 mM into lungs. Collected bronchoalveolar lavage fluid (BALF) was centrifuged 10 min at 110 g and supernatants were stored at −80°C for further analyses.

Immunohistochemistry
Details are provided in the online supplementary material and methods section.

Statistical analysis
All statistical analyses were performed using GraphPad Prism software. Results were expressed as means ± SEM. An unpaired t-test was used to compare two groups with normally distributed data. When the assumption of normality was not met, the non-parametric Mann–Whitney test was used instead. For comparisons of means between more than two experimental groups, one-way analysis of variance (ANOVA) was used as appropriate. P value > 0,05 was considered not significant (ns); P values are provided within each figure legend, together with the statistical test performed for each experiment.

Results

Chronic exposure to DEP favours tumour progression in a genetically engineered mouse model of lung adenocarcinoma

To assess the influence of DEP exposure on tumour development, we used a genetically engineered mouse model that develops lung tumours upon KRAS oncogenic activation combined with p53 loss (KP model). Tumour burden in the lungs of KP mice was assessed 90 days after AAV-Cre administration (Fig 1A).
Strikingly, we observed larger tumour nodules in DEP-exposed mice (Fig. 1B and C). Additionally, the density of Ki67+ cells within tumour nodules was higher in DEP-exposed KP mice, indicating increased tumour cell proliferation in the DEP-altered lung microenvironment (Fig. 1D and E).
Although total CD4+ and CD8+ T cell numbers were unchanged in the lung following DEP chronic exposure (Fig. 1H, 1L), we observed alterations in the composition and activation state of T cell populations. Specifically, there was an increase in Treg numbers (Fig. 1I), along with an upregulation of activation and exhaustion markers such as CD69, PD-1 and TIM3, as well as CD39, which contributes along with CD73 to adenosine production in the lung microenvironment, thereby promoting immunosuppression (Fig. 1F, 1G, 1J, 1K). Of note, expression of these makers on CD8+ T cells was less compared to CD4+ T cells.
While we observed no changes in the total counts of monocytes (Ly6ChighLy6G-) upon chronic DEP exposure in our KP model (Fig. 1N), we did observe a markedly increase in CD14pos PMNs, a subset typically associated with PMN-MDSCs (Fig. 1O). In contrast, no significant differences were observed in the total numbers of CD14neg PMNs.

Acute exposure to DEP induces CD14pos recruitment and NET formation in the lungs
To further explore the underlying mechanisms and to dissect the specific immune alterations by which DEP exposure influence lung tumour development, we first assessed lung inflammation caused by acute DEP exposure in C57BL/6 mice (Fig. 2A) by quantifying lung immune cell populations by flow cytometry. Acute DEP exposure led to a significant infiltration of polymorphonuclear neutrophils (PMNs) and a reduction in alveolar macrophages (AMs), while monocytes (MOs), interstitial macrophages (IMs), eosinophils, conventional dendritic cells (cDCs), CD4+ and CD8+ lymphocytes remained unchanged (Fig. 2B). Interestingly, while monocyte numbers remained unaffected (Fig 2C, 2E), we observed that acute DEP exposure was sufficient to induce the emergence of a CD14pos PMN subpopulation (Fig. 2C-F). Of note, the emergence of this population was restricted to the local lung environment, since no presence of CD14pos PMNs was detected in the spleen despite the overall increase in PMNs following DEP exposure. (Fig. S4A, S4C).
Considering the established link between NET formation and tumour progression [4,9], we investigated whether PMNs increase NET formation after DEP acute exposure. We identified NETs in lung histological samples by the co-localization of citrullinated-histone (Cit-H3), myeloperoxidase (MPO), and DNA -key components of neutrophil granules that are released during NET formation. NET formation was observed in lung tissue of mice exposed to DEP administration but was absent in control mice (Fig. 2G). Furthermore, mice exposed to DEP showed significantly higher levels of dsDNA in their BALF as compared to the control group (Fig. 2H). These results demonstrate that acute DEP exposure triggers the recruitment of PMNs into the lung, promotes the appearance of a CD14pos subpopulation, and leads to NET formation.

DEP-recruited CD14pos PMNs exhibit an immunosuppressive phenotype
To elucidate the functional immunosuppressive characteristics of PMNs recruited to the lungs in acute DEP-exposed mice (Fig. 3A), we isolated PMNs based on CD14 expression (Fig. 3B). No distinct morphological differences were observed between CD14neg and CD14pos PMN populations (Fig. 3C). However, immunohistochemistry analysis revealed increased NET formation in CD14pos PMNs compared to their CD14neg counterparts (Figs. 3D, 3E). Next, we quantified in both PMN populations the expression of key genes associated with the immunosuppressive activity of MDSCs, such as Arg1, Nos2, Cd274, Ptgs2, Il-10 and Tgfβ [7]. RT-qPCR analysis demonstrated a significant upregulation of all assessed genes in CD14pos PMNs compared to CD14neg PMNs in DEP-exposed mice (Fig. 3F). In addition, CD14pos PMNs also exhibited elevated protein expression of Siglec-F, PD-L1 and Arg1 compared to CD14neg PMNs (Fig. 3G).
To functionally confirm the suppressive activity of DEP-recruited CD14pos PMNs, we conducted a CD8+
T cell suppression assay. Both CD14neg and CD14pos PMNs isolated from DEP exposed lungs were cultured in presence of activated T cells isolated from lymph nodes of naïve mice (Fig. 3H). The CD14neg PMNs exhibited no detectable suppressive activity when co-cultured with activated T cells, whereas co-culture with CD14pos PMNs significantly reduced T cell proliferation (Fig. 3I, 3J). These results demonstrate that CD14pos PMNs exhibit a MDSC-like phenotype and are functionally able to suppress T cell proliferation, potentially contributing to the establishment of an immunosuppressive microenvironment in the lungs of DEP-exposed mice.

Chronic exposure to DEP induces an immunosuppressive microenvironment in the lung
We next investigated whether chronic DEP exposure, which may better reflect real-world environmental conditions, also promotes the emergence of CD14pos PMNs with immunosuppressive properties in the lung (Fig. 4A). Consistent with our observations in the acute exposure model, DEP-exposed lungs showed a significant increase in numbers of PMNs compared to controls, along with a higher proportion of CD14pos PMNs (Fig. 4B, 4D), while monocytes remained unchanged (Fig. 4B-C). Furthermore, CD14pos PMNs exhibited significantly higher expression of Siglec-F and PD-L1, as well as regulatory markers, such as CD73, which is involved in adenosine production [10], and SIRPα, which is involved in the inhibition of phagocytosis of CD47-expressing target cells [11], compared to CD14neg PMNs (Fig. 4E). In the spleen, on the other hand, no significant differences in PMN recruitment were observed and CD14pos PMNs were not detected in either group (Fig. S5 A-B).
Although total CD4+ and CD8+
T cell numbers again remained unchanged in the lung following DEP chronic exposure (Fig. 4G, 4K), we observed alterations in the composition and activation state of T cell populations. Specifically, there was an increased proportion of Tregs (Fig. 4I), along with an upregulation of activation and exhaustion markers such as CD69, PD-1, LAG-3 (only in CD4+ T cells) and CD39. Similar to what we observed in the KP model, these alterations appear to be more pronounced in CD4⁺ T cells than in their CD8⁺ counterparts (Fig. 4F, 4H, 4J, 4L). These results demonstrate that chronic DEP exposure induces the emergence of MDSCs-like PMNs with a regulatory phenotype that may promote immunosuppression in the context of cancer, given the pivotal roles of PD-L1, CD73 and SIRPα in suppressing anti-tumour immune responses [[10], [11], [12]]. Furthermore, we observed that this regulatory and immunosuppressive signature extended to T cell populations, which showed increased expression of regulatory and activation/exhausted markers, changes that may impair effective T cell responses.

Discussion
In this study, we investigated the mechanisms by which exposure to DEP may promote lung cancer progression. DEP are commonly found in cities, in places where diesel engines are used indoors or along roads. Previous studies have suggested that engine exhaust contributes to the development of lung tumours by inducing oxidative stress, inflammation and genotoxic effects [13,14]. However, the impact of DEP exposure on anti-tumour immune mechanisms remains elusive to date, and a comprehensive understanding of its effects on anti-tumour immunity is lacking.
Consistent with previous reports, our study supports the notion that exposure to DEP induces neutrophilic inflammation in the lungs, a phenomenon that has been directly linked to the promotion of lung metastasis [15]. Interestingly, similar mechanisms have been described in models of ozone exposure, where neutrophilic infiltration was also associated with enhanced metastatic dissemination [9]. Our results provide critical insights into the immunosuppressive signature of PMNs, particularly CD14pos PMNs, following acute and chronic DEP exposure. CD14pos PMNs in the lungs formed NETs as confirmed by the co-localization of citrullinated histone (Cit-H3) and extracellular DNA. NETs can contribute to tumour progression in multiple ways. Firstly, NETs are known to entrap circulating tumour cells and facilitate lung tissue invasion [9,16]. Furthermore, NETs can also suppress anti-tumour immune responses by coating tumour cells, thereby protecting them against cytotoxic CD8+
T cells and NK lymphocytes [17]. In addition, NETs can directly interact with infiltrating T cells by the expression of PD-L1 which engages PD-1 receptors on T cells. Such interaction promotes the upregulation of multiple inhibitory receptors, leading to diminished mitochondrial function and a shift toward a metabolically exhausted state [18].
Besides undergoing NETosis, DEP-recruited CD14pos PMNs expressed key MDSC-associated genes, including Arg1, Nos2, Ptgs2, and Cd274 [19]. In addition, compared to their CD14neg counterparts, CD14pos PMNs exhibited increased expression of immunoregulatory and immunosuppressive cytokine such as IL-10 and TGFβ, along with Siglec-F expression, a marker associated with neutrophils displaying MDSC-like features [20]. CD14pos PMNs demonstrated a functional ability to suppress T cell proliferation, which aligns with prior studies showing that these cells can promote immune suppression in cancer models, facilitating immune evasion and reducing anti-tumour responses [21]. The presence of CD14pos PMNs in both cancer and non-cancer inflammatory conditions suggests that CD14 expression on tumour-infiltrating neutrophils reflects a broader inflammation-associated phenotype. These findings indicate that DEP-recruited CD14pos PMNs acquire an immunosuppressive phenotype, combining both molecular and functional characteristics of PMN-MDSCs, which likely contribute to the establishment of a tumour-permissive immune microenvironment.
T cell exhaustion is a state of T cell dysfunction commonly observed in chronic infections and cancer. Exhausted T cells exhibit overexpression of inhibitory receptors and decreased effector cytokine production, resulting in a failure to eliminate cancer cells [22]. In the current study, chronic DEP led to the upregulation of inhibitory receptors commonly associated with an exhausted T cell phenotype in chronic settings, such as PD-1, LAG-3 and TIM-3, suggesting that chronic exposure to pollutants may drive T cell dysfunction [23,24]. Furthermore, other markers involved in immunosuppression, such as CD39, was also upregulated on T cells, along with CD73 on CD14pos PMNs. CD39 and CD73 function together to convert extracellular ATP to adenosine, a molecule that inhibits T cell function and promotes Treg activity [25]. In line with this, the numbers of Tregs increased in the lungs of DEP-exposed mice.
Our findings, together with those of Hill et al., provide compelling evidence that airborne pollutants play a crucial role in lung cancer progression by shaping the tumour microenvironment rather than directly inducing mutations. Hill et al. demonstrated that PM2.5 exposure promotes EGFR- and KRAS-driven lung adenocarcinoma through an inflammatory response characterized by sustained influx of interstitial macrophages which upregulate PD-L1 and produce IL-1β. This pro-inflammatory environment facilitates the expansion of pre-existing oncogenic clones and reprograms EGFR -mutant alveolar type II (AT2) epithelial cells into a progenitor cell state, fostering tumour initiation and progression [26]. Our study extends these findings by showing that chronic DEP exposure promotes an immunosuppressive tumour milieu in KP mice, characterized by the appearance of immunosuppressive PMNs and the emergence of a dysfunctional T cell state.
Taken together, these findings underscore the multifaceted role of air pollution in lung cancer progression, involving both inflammatory and immunosuppressive pathways. Overall, our study highlights the impact of DEP on lung inflammation and its role in establishing an immunosuppressive, tumour-promoting lung microenvironment. Patients chronically exposed to high levels of PM may develop a tumour microenvironment enriched in immunosuppressive marks. As such, they could particularly benefit from immunotherapy targeting the CD47-SIRPα axis and CD73 molecules expressed on immunosuppressive neutrophils. In addition, the use of well-established immune checkpoint inhibitors, such as PD-1/PD-L1 blockers or LAG-3 antagonists, may hold enhanced therapeutic relevance in lung cancer patients exposed to high levels of air pollution. These approaches offer promise to counteract the immunosuppressive effects of DEP exposure and to restore effective anti-tumour immune responses.

Funding sources
This work was funded by the FRS-FNRS (grant T.0036.25 and Télévie 7.6504.24) (Belgium), the Foundation against cancer (PDR grant 2024.187) (Belgium), the Fondation Léon Fredericq (University of Liege, Belgium), the Fonds spéciaux of the University of Liège (Belgium), the European Regional Development Fund (FEDER) - SYST-IMM project.

CRediT authorship contribution statement
Marie-Laure Delhez: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing. Maëlle Bosmans: Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. Lucia Rodriguez Rodriguez: Methodology, Validation, Writing – review & editing. Alison Gillard: Conceptualization, Investigation, Methodology. Silvia Blacher: Data curation, Formal analysis, Methodology. Arnaud Blomme: Investigation, Methodology, Writing – review & editing. Pierre Close: Methodology, Writing – review & editing. Bénédicte Machiels: Formal analysis, Methodology, Writing – review & editing. Marie-Julie Nokin: Formal analysis, Investigation, Methodology, Supervision, Validation, Writing – review & editing. Didier Cataldo: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.