Background The process of epithelial-mesenchymal transition (EMT) has been regarded in systemic sclerosis (SSc) as one of the possible mechanisms favouring tissue accumulation of monocyte-derived fibrocytes or myofibroblasts, which contribute to tissue fibrosis . Forkhead box E3 (FOXE3) is a transcription factor involved in EMT of lens epithelial cells (LEC). Its expression progressively decreases with the migration of LEC from the anterior to the equatorial region. FOXE3 expression cessation marks initiation of fiber differentiation, suggesting that the loss of FOXE3 expression favors a pro-fibrotic phenotype . No data are available on mRNA FOXE3 expression in sites other than LEC.
Objectives In this study, we investigated the FOXE3 mRNA expression in unstimulated and TGF-β- or IL-4-stimulated monocytes from SSc patients and healthy blood donors (HBD), to established whether i) FOXE3 is constitutively expressed in human monocytes; ii) FOXE3 expression can be modulated in vitro by cytokines involved in SSc profibrotic process; iii) there is any association between FOXE3 expression and a particular SSc serological profile.
Methods PBMC were isolated from heparinized peripheral blood of 9 patients with SSc (5 Scl70+; 4 Scl70–), and 3 HBD by Ficoll-Hypaque density gradient centrifugation. Monocytes (CD14+) were isolated by positive selection using microbeads. Cells (1x106 cells/ml) were stimulated TGF-β (10 ng/ml) and IL-4 (40 ng/ml) for 14 days. mRNA was extracted and semi-quantitative PCR was performed to assess FOXE3 expression. GM-CSF stimulation (50ng/ml) was used as positive control. The levels of FOXE3 mRNA were quantified by normalizing its expression against that of GAPDH. Expression was measured as mean relative expression level (MREL). Variation of expression was measured as mean fold change (MFC).
Results Similar baseline levels of FOXE3 mRNA was observed in unstimulated CD14+ cells from SSc patients and HBD (MREL SSc=0.32; HBD=0.26). As expected, GM-CSF stimulation of CD14+ cells from SSc patients and HBD markedly up-regulated FOXE3 expression (SSc: MFC=3.24; HBD: MFC=1.84). TGF-β and IL-4 behaved similarly to GM-CSF in enhancing FOXE3 expression in CD14+ cells from all HBD (MFCTGF-β=1.35; MFCIL-4=1.59) and from 3 out 4 Scl70– patients (MFCTGF-β=2.36; MFCIL-4=2.9), being the expression unchanged in the remaining Scl70– patient. By contrast, in the 4 Scl70+ patients, CD14+FOXE3 expression markedly decreased following these cytokines stimulation (MFCTGF-β=0.28; MFCIL-4=0.31)
Conclusions This is the first study to demonstrate FOXE3 mRNA expression in monocytes from HBD and SSc patients, and its differential expression following TGF-β and IL-4 stimulation, correlating with the serological profile of SSc patients. The data suggest that the down-regulation of FOXE3 induced by TGF-β and IL-4 may direct monocytes toward a more profibrotic phenotype in Scl70+ as compared to Scl70– patients. The relationship of this finding with the anti-FOXE3 antibodies recently detected in SSc sera , remains to be determined.
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Acknowledgements This work was supported by a 2013 grant from the Italian Group for Systemic Sclerosis (GILS), Milan, Italy.
Disclosure of Interest None declared