Background/Purpose: Anti-citrullinated peptide/protein antibodies (ACPA) may be detected in RA serum more than 10 years before onset of disease, suggesting that the autoimmune response begins outside the joint. Because smoking and periodontitis are risk factors for RA, it has been suggested that smoke inhalation or invasive infection with Pophyromonas gingivalis leads to citrullination of proteins in the lung. In this study, we examine human lung tissue by immunoblotting from 41 subjects with well-defined smoking status for the presence of citrullinated proteins, PAD2, PAD4 and P. gingivalis DNA.

Methods:

Uninvolved tissue samples were dissected from lobectomy specimens from 41 subjects (10 never smokers, 10 COPD ex-smokers, 10 smokers without airflow limitation and 11 COPD smokers). The tissue was homogenised and examined by immunoblotting for the presence of citrullinated proteins with AMC. PAD2, PAD4, α-enolase, citrullinated α-enolase, vimentin and fibrinogen were detected using specific antibodies. Band intensities were scored quantitatively using a BioRad Quantity One Analysis Sofware or semi-quantitatively from 0-3 by two blinded observers. Recombinant proteins were used as positive controls and blots with secondary antibodies alone were carried out to exclude non-specific cross-reactivity.  P. gingivalis was detected by nested PCR using primers spanning the PPAD gene.

Results:

Citrullinated proteins were found in all lung tissues, both smokers and non-smokers. There was a trend towards increased citrullination in the lungs of the smokers with and without COPD but this did not reach statistical significance by either densitometry or semi-quantitative scoring of band intensities. There was a similar, but not significant, trend for increased expression of PAD2 amongst the smokers. PAD4 and the RA antigens, α-enolase, vimentin and fibrinogen, were observed in all lung tissue in comparable amounts regardless of disease and smoking status. There was also evidence of citrullination of α-enolase provided by co-migration of a ~50kDa band recognised by AMC and an anti-citrullinated enolase antibody, and by 2D electrophoresis showing multiple isoforms of the antigen.

P. gingivalis DNA was detected most frequently in all the never smokers (100%) compared to the COPD ex-smokers (10%); smokers without COPD (10%); and least frequently in COPD smokers (0%) (Chi-square test, p<0.001).

Conclusion: We have demonstrated widespread citrullination of proteins in lung tissue from never smokers, with a slight increase with smokers. The inverse relationship between smoking and the detection of P. gingivalis DNA was initially surprising, but in keeping with previously published studies. These data support the hypothesis that the lung is a site for priming the ACPA response, though the mechanism is likely to be more complex than smoking inducing PAD enzymes or P. gingivalis infection.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/influence-of-smoking-on-citrullinated-proteins-and-porphyromonas-gingivalis-in-the-lung-a-priming-site-for-autoimmunity-in-ra/