PM10 collection and stock preparation

Coarse particulate matter (PM10) was collected in Valle de Aburrá, Colombia, and obtained as previously described [23]. Briefly, 100 quartz filters were obtained and cut into small pieces, and stock suspensions were prepared in high-purity sterile water by sonication followed by lyophilization. To perform the assays, the PM10 stimuli were prepared in sterile water and stored at -20 °C until use.

Isolation and stimulation of peripheral mononuclear blood cells (PBMCs)

Peripheral blood samples were obtained from healthy adult donors (males and females between 21 and 56 years old), with no history of smoking or pre-existing inflammatory conditions such as allergies or autoimmune diseases. In addition, individuals with symptoms of infectious diseases within weeks prior to sampling were excluded. Ficol-histopaque 1077 (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was used to isolate the PBMCs by gradient method. Then, PBMCs were seeded at a density of 250,000–2,000,000 in RPMI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin (all reagents from Sigma-Aldrich). Cells were exposed to PM10 (1–400 µg/mL) and incubated for 2–24 h (to evaluate the best timing for gene expression of inflammatory mediators) at 37 °C with 5% CO2. As a positive control for cytotoxicity, Triton X-100 (5%) was used, while LPS (0.05 µg/mL; Sigma-Aldrich L4391-1MG) was used for cytokine assays. The cellular fractions and supernatants were collected by centrifuging at 730 x g for 10 min and stored at -20 °C until further use.

Cytotoxicity assessed by lactate dehydrogenase (LDH)

Cell cytotoxicity was evaluated by the LDH colorimetric assay CyQUANT™ Kit (Invitrogen, Waltham, Massachusetts, USA) after 24 h of stimulation, following the manufacturer’s instructions. The reading was performed in a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific, Waltham, Massachusetts, USA) at 490 nm.

Inhibition assay

The PBMCs were seeded at a density of 250,000 cells/well in RPMI-1640 medium (2% FBS, 1% penicillin-streptomycin and 2 mM L-glutamine) and treated with glibenclamide or curcumin (commercial formulas, Merck Laboratory and Coaspharma Laboratory, respectively) for 1 h at 37 °C with 5% CO2. Then, the PBMCs were exposed to PM10 (50 and 100 µg/mL) and incubated overnight. Cell supernatants were collected for ELISA.

RNA extraction and cDNA synthesis

Total RNA was extracted from 2,000,000 cells/well with the Direct-zol RNA Miniprep kit (Zymo Research), according to the manufacturer’s recommendations. RNA purity and quantity were assessed by spectrophotometry at 260–280 nm (Nanodrop one, Thermo Fisher Scientific, USA). From 150ng of total RNA, cDNA was constructed using the iScript cDNA synthesis kit (Bio-Rad, USA).

Real-time PCR (qPCR)

qPCR was performed to quantify the mRNA of the inflammatory response components, using SYBR Green qPCR Master mix kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported [24]. Briefly, the qPCR conditions were standardized for each gene following an amplification protocol of 40 cycles (Table S1). The relative mRNA content of NLRP3, NLRP1 (NLR Family Pyrin Domain Containing 1), NLRC4 (NLR Family CARD Domain Containing 4), AIM2 (Absent In Melanoma 2), TXNIP (Thioredoxin Interacting Protein), ASC, Caspase-1, Caspase-5, IL-1β, IL-18, IL-36γ, IL-6, IL-8 and TNF-α were quantified; Phosphoglycerate kinase (PGK) gene expression was used to normalize the RNA content. For qPCR analysis, CFX Manager Version: 1.5.534.0511 software (Bio-Rad, Hercules, CA, USA) was used, and the ΔΔCt method was applied to calculate the relative gene expression levels.

Cytokine quantification by ELISA

The PBMC supernatants were used to quantified IL-1β, IL-6, IL-8 and TNF-α levels after 24 h of PM10 exposure, by ELISA (#437,004, Biolegend, Thermo Fisher; #555,220, BD Biosciences, San Jose, CA, USA; #431,504 Biolegend, Thermo Fisher, and #88-7346-76 eBioscience, Thermo Fisher Scientific, respectively) as previously reported [24].

ASC complex formation

THP-1 cells expressing green fluorescent protein (GFP) coupled to the ASC protein (THP-1-ASC-GFP) were seeded at a density of 10,000 cells/well in 96 well-plates, and using Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) with 10% of FBS for 24 h at 37 °C with 5% CO2. THP-1-ASC-GFP cells were primed with LPS (0.1 µg/mL) overnight and then exposed to increasing doses of PM10 for 1 h, 3 h and 5 h. Visualization was performed by confocal microscopy.

in vivo model

Eight-week-old male C57BL6 mice obtained from Charles Rivers (Portage, MI, USA) were bred and housed at 22 ± 1 °C, with free access to food and water and maintained under a 12 h light/dark cycle. The in vivo assays were performed under specific pathogen-free conditions at the Universidad de Antioquia animal facility (Medellín, Colombia). Mice were treated daily intranasally with PM10 (10 and 100 µg/mL in PBS) or vehicle (PBS) for 5 days; for each dose, the final volume was 50µL. On day 6, the mice were euthanized via intraperitoneal overdose of ketamine/xylizane (100/10 mg/kg). In total, eighteen mice were included, six mice per treatment (PM10 or PBS).

The weights of the mice were monitored every day. After euthanasia, a cardiac puncture was performed to collect whole blood, and BALF was obtained by perfusion of the lungs with 1 mL of sterile PBS. Total cell count and differential count were performed by light microscopy and Wright’s stain, respectively, from BALF. For histopathological analysis, the left lobe of the lungs was fixed with paraformaldehyde for 48 h, embedded in paraffin, and stained with hematoxylin-eosin (Sigma Aldrich).

The lung’s right lobe and peribronchial nodule were lysed in TRIzol-Reagent. RNA extraction was performed with Direct-zol RNA Miniprep kit (Zymo Research, Orange, CA, USA), according to the manufacturer’s instructions. RNA quantification, cDNA construction and qPCR were performed as described in the previous section to quantify NLRP3, Caspase-1, IL-1β, IL-18, IL-6, CXCL1 and MUC5AC. Additionally, IL-6 levels in BALF and serum were quantified using ELISA kits (#431,301, Biolegend, Thermo Fisher).

Statistical analysis

The data analysis was performed using GraphPad Prism 9.0.2 (GraphPad Software, CA, USA). Normality and homoscedasticity were assessed using the Shapiro-Wilk and Levene tests, respectively. Comparisons between two or more groups were made using parametric ANOVA or Kruskal-Wallis tests with a 95% confidence. In case of statistical differences, the post hoc tests HDS of Tukey and Dunn, respectively, were applied. Significant differences were considered with p-values were lower than 0.05. The data are presented as the median ± IQR (interquartile range).

Ethics

All the participants enrolled in the study were adults, who read and signed an informed consent form. Te study was previously reviewed and approved by the research ethics committee from Universidad Cooperativa de Colombia (Act 003 / 2018). Additionally, the experiments were carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The animal experiments were carried out following international guidelines and by the animal ethics committee from Universidad de Antioquia (Act 117 / 2018).