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. 2017 Dec 29;18(1):218.
doi: 10.1186/s12931-017-0692-9.

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis

Katerina D Samara  1   2 , Athina Trachalaki  1 , Eliza Tsitoura  1 , Anastasios V Koutsopoulos  3 , Eleni D Lagoudaki  3 , Ismini Lasithiotaki  1 , George Margaritopoulos  2 , Panagiotis Pantelidis  2 , Eleni Bibaki  1 , Nikolaos M Siafakas  1 , Nikolaos Tzanakis  1 , Athol U Wells  2 , Katerina M Antoniou  4
Affiliations

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis

Katerina D Samara et al. Respir Res. .

Abstract

Background: Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear.

Methods: We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified.

Results: PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs).

Conclusions: These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.

Keywords: Citrullination; Idiopathic pulmonary fibrosis; Interstitial lung disease; Pathogenesis; Peptidylarginine deiminases; Rheumatoid arthritis; Smoking.

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Conflict of interest statement

Authors’ information

"Dr Katerina Samara is the recipient of a European Respiratory Society Fellowship (STRTF 27-2010)".

Ethics approval and consent to participate

Informed consent was obtained from all patients who participated in this study. The study was approved by the Ethics Committees of the University Hospital of Heraklion (IRB number: 1045 and17030) and the Royal Brompton Hospital (REC reference 13/LO/0857).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
(a) PADI4 and (b) PADI2 relative mRNA expression in IPF, RA-ILD and control subjects were analysed by RT-PCR and normalised to actin. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, Mann-Whitney test
Fig. 2
Fig. 2
a) A representative immunoblot of PADI4 intracellular levels in 3 controls, 4 IPF and 3 RA-ILD subjects. b) PADI4 protein expression levels in IPF, RA-ILD and control subjects relative to actin. The bars represent median with range. ** = p < 0.01, Mann-Whitney test
Fig. 3
Fig. 3
Representative images of BALF cytospins, stained with anti-PADI4 antibody, from IPF (a) and RA-ILD (b) patients, demonstrated strong cytoplasmic staining mostly in polymorphonuclear leukocytes (arrows) and mild stain in some macrophages (arrowheads), whereas in some IPF (c) and control patients (d) staining was observed mostly in macrophages (arrows) and only in occasional granulocytes (arrowheads). Negative control (e). Magnification X600
Fig. 4
Fig. 4
a) A representative immunoblot of citrullinated proteins in four (4) IPF, five (5) RA-ILD and two (2) control subjects. b) Total citrullinated proteins in IPF, RA-ILD and control subjects relative to actin. The bars represent mean and SD. * = p < 0.05, t-test with Welch correction, kDa: kilodaltons
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