Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which loss of tolerance to citrullinated proteins is considered an early pathogenic event [1]. While the lower airways have been implicated as a site of mucosal citrullination, the upper airway epithelium—highly exposed to inhaled environmental triggers—remains insufficiently characterized. Airborne particulate matter (PM) has been associated with increased RA risk, suggesting that pollutant-driven epithelial stress may promote PAD-mediated protein citrullination and local immune activation. Studies further indicate that long-term exposure to specific PM ₂ . ₅ components (e.g., black carbon) may contribute to immune dysregulation and has been linked to increased risk of arthritis and RA [2,3].Overall, although direct causal evidence connecting PM exposure to citrullination in upper airway epithelial cells is limited, the inflammatory inducing properties of PM may plausibly facilitate processes that promote citrullination and autoimmune responses in individuals with RA [4–8].

Objectives: The objective of this research is to determine whether citrullinated proteins are detectable in nasal swabs from RA patients compared with healthy controls, and whether exposure to airborne particulate pollutants (PM ₂ . ₅ and PM ₁₀ ) induces PADI(1-4,6) gene expression in the upper airway epithelium.

Methods: Primary human nasal epithelial cells (HNEC) and PM stimulation: Primary human nasal epithelial cells (HNEC) (n = 8; RA n = 4, healthy controls (HC) n = 4; 6F/2M) were collected by gentle brushing of the middle meatus using cytology brushes, isolated, and cultured on collagen-coated plates. All RA patients fulfilled the ACR/EULAR 2010 classification criteria. HNEC were cultured in monolayer and stimulated for 24 h with 80 µg/mL PM ₂ . ₅ or PM ₁₀ using JRC reference materials ERM-CZ110 and ERM-CZ120, respectively. Cell viability was assessed using a resazurin assay, and a dose–response curve was generated to define IC ₂₀ for PM ₂ . ₅ (the most cytotoxic fraction), which guided exposure conditions for downstream analyses.

PADI gene expression: Gene expression was assessed using TaqMan assays and normalized to GAPDH and ACTB (housekeeping genes). Changes in PADI gene expression ( PADI1–4 and 6 ) were calculated relative to unstimulated controls using the ΔΔCt method and reported as 2^ ^-ΔΔCt .

Citrullinated proteins in nasal swabs (ELISA): Citrullinated proteins were quantified in nasal swabs using a commercial ELISA according to manufacture protocol in a pilot cohort (n = 29; RA n = 16, HC n = 13). Nasal swabs provide an ex vivo readout and reflect the nasal mucosa as a first-line, chronically exposed interface to environmental factors.

Statistics: Statistical analyses were performed in GraphPad Prism 10. Normality was assessed using the Shapiro–Wilk test. Group comparisons were performed using the Mann–Whitney test.

Results: In the pilot cohort, nasal swabs from RA patients showed higher levels of citrullinated proteins than those from healthy controls (median HC vs RA: 3.14 vs 5.24 ng/mL, p = 0.012), suggesting that citrullination processes may occur locally in the nasal mucosa and are enhanced in RA patients. Under the selected exposure conditions, both PM ₂ . ₅ and PM ₁₀ modulated PADI gene expression in primary nasal epithelial cells, with increased expression observed for PADI1 (fold change vs unstimulated: PM ₂ . ₅ : HC 1.70, p = 0.004; RA 1.83, p = 0.012; PM ₁₀ : HC 1.36, p = 0.004; RA 1.41, p = 0.004) and PADI2 (fold change vs unstimulated: PM ₂ . ₅ : HC 1.15, p = 0.016; RA 2.17, p = 0.012; PM ₁₀ : HC 1.26, p > 0.050; RA 1.38, p = 0.004) compared with unstimulated cells. These findings suggest that PM exposure may modulate PAD-related gene expression in the nasal epithelium. Although preliminary and based on a limited sample size, these data support the concept of local mucosal citrullination in RA.

Conclusions: Together, the results support the hypothesis that the nasal mucosa as first-line of contact with inhaled particles may represent a relevant upper-airway site for local protein citrullination in RA. Airborne particulate matter can influence PAD-associated gene expression in primary nasal epithelial cells in both RA and HC and citrullinated proteins are increased in nasal swabs from RA patients compared with healthy controls. However this is a pilot cohort study and larger cohorts and mechanistic follow-up studies are warranted to define PAD activity, identify specific citrullinated targets, and clarify how pollutant-induced epithelial stress may contribute to early mucosal immune dysregulation and RA-related autoimmunity.

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Acknowledgments: NIL.

Disclosure of Interests: None declared.