EXPRESSION OF THE TRANSCRIPTION FACTOR FORKHEAD BOX E3 (FOXE3) IN MONOCYTES FROM PATIENTS WITH SYSTEMIC SCLEROSIS AND CORRELATION WITH THEIR SEROLOGICAL PROFILE

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 [1]. 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 [2]. 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_ß_ 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 (1x10 6 cells/ml) were stimulated TGF_ß (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_ß and IL_4 behaved similarly to GM_CSF in enhancing FOXE3 expression in CD14 + cells from all HBD (MFC TGF_ß =1.35; MFC IL_4 =1.59) and from 3 out 4 Scl70 – patients (MFC TGF_ß =2.36; MFC IL_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 (MFC TGF_ ß =0.28; MFC IL_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_ß and IL_4 stimulation, correlating with the serological profile of SSc patients. The data suggest that the down_regulation of FOXE3 induced by TGF_ß 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 [3], remains to be determined. References: Postlethwaite AE et al. Curr Opin Rheumatol 16:733_738, 2004. Landgren H et al. Invest Ophthalmol Vis Sci 49:4269_4277, 2008. Perosa F et al. Arthritis Res Ther 15:R72, 2013. Acknowledgements: This work was supported by a 2013 grant from the Italian Group for Systemic Sclerosis (GILS), Milan, Italy. Disclosure of Interest: None declared DOI: 10.1136/annrheumdis_2014_eular.4130 Citation: Ann Rheum Dis 2014;73(Suppl2

Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, and as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model

Autoantibodies Recognizing the Amino Terminal 1-17 Segment of CENP-A Display Unique Specificities in Systemic Sclerosis.

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, and as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model