In central Pennsylvania, USA, and local communities, 81 individuals were recruited. Controls have no history of welding, whereas welders were defined as persons who have welded at any point in their lifetime. All individuals were male. Written informed consent was obtained in accordance with guidelines approved by the Penn State Health Milton S. Hershey Institutional Review Board. Controls were age-matched volunteers from the same regional community with various occupations. Before sending the serum samples to Iowa state, samples were blinded. This serum was used to isolate exosomes as described. Of the exosome samples, 39 samples were chosen randomly for further Western blot, qRT-PCR, and Luminex analysis.

Quantitative reverse transcription polymerase chain reaction

RNA was isolated from tissues and cells according to a previous publication (98, 99). Briefly, cells or tissues were lysed in 1 ml of TRIzol reagent and incubated for 5 min. After incubation, 0.2 ml of chloroform was added to each tube, incubated for 2 min, and centrifuged at 12,000g for 15 min at 4°C. After centrifugation, the top clear layer containing RNA was transferred to a fresh tube containing 0.7 ml of isopropanol, incubated for 15 min, and centrifuged at 12,000g for 10 min at 4°C to precipitate the RNA. After precipitation, the supernatant was discarded, and the pellet was washed with 75% ethanol, air-dried, and dissolved in water. A NanoDrop was used to quantify the RNA, and 1 μg of RNA was used for converting into complementary DNA (cDNA). For cDNA synthesis, the high-capacity cDNA synthesis kit from Applied Biosystems (catalog number 4368814) was used according to the manufacturer’s protocol. Quantitative SYBR green PCR assay was performed using qRT2PCR SYBR Green Mastermix (Agilent) and prevalidated primers. The validated primers used from QIAGEN were the following: pro–IL-1β (QT01048355), NLRP3 (QT00122458), NLRC4 (QT00264670), AIM2 (QT00266819), Nos2 (QT00100275), Mfn2 (QT00134295), Mul1 (QT00132734), SLC30A10 (QT01199009), SLC11A2 (QT01047368), VPS35 (QT00160258), VPS29 (QT00137228), and 18S (QT02448075). The fold change in gene expression was determined by the ΔΔCt method, where Ct is the threshold value. 18S was used as the housekeeping gene.

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