Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus

Abstract

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10-4) and 60% versus 28% (P = 3.10-8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10-10) and 82% (P = 5.10-13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

Fig 1. Patients with scleroderma and patients with lupus have autoantibodies (AAb) against A) EphB2 and B) THEX1 proteins.

Under our ELISA conditions, both proteins are significantly recognized by sera from patients with systemic sclerosis (SSc) and patients with Systemic Lupus Erythematosus (SLE), when compared to both sera from healthy controls (HC) and sera from patients with other rheumatic diseases (RD). All p values (P) are calculated after Bonferroni correction. P₁: patients with SSc or SLE compared to HC, P₂: compared to RD, P₃: compared to both HC and RD.

Fig 2. Autoantibodies against A) EphB2 and B) THEX1 analyzed in patients with scleroderma.

Sera reactivity against A) EphB2 or B) THEX1 is given by Absorbance (Abs) value for patients with systemic sclerosis (SSc), patients with SSc positive for anti-topoisomerase antibody (ATA^pos), positive for anti-centromere antibody (ACA^pos) or negative for both (ACA/ATA^neg), and compared to all controls (CTLS) including healthy controls (HC) and controls with other Rheumatic diseases (RD), with a total of n = 166 with 85 HC and 81 RD for EphB2 and n = 180 with 99 HC and 81 RD for THEX1. Sera were tested at dilution 1/100 and defined as positive when Abs≥0 (on or above the dotted blue line). Red bars represent medians with interquartile ranges. Percentage of individuals positive for anti-EphB2 or THEX1 antibodies are indicated in the upper part of the graph. All P values are calculated using Mann Whitney test by comparing Abs results from patients with SSc as a whole group or in SSc subgroups to all controls (CTLS).

Fig 3. Autoantibodies against A) EphB2 and B) THEX1 analyzed in subgroups of patients with lupus.

Sera reactivity against EphB2 or THEX1 is given by Absorbance (Abs) value for patients with Systemic Lupus Erythematosus (SLE), positive for anti-dsDNA antibodies (dsDNA^pos) or negative (dsDNA^neg) and compared to all controls (CTLS) including healthy controls (HC) and controls with other Rheumatic diseases (RD), with a total of n = 166 with 85 HC and 81 RD for EphB2 and n = 180 with 99 HC and 81 RD for THEX1. Sera were tested at dilution 1/100 and defined as positive when Abs≥0 (on or above the dotted blue line). Red bars represent medians with interquartile ranges. Percentage of individuals positive for anti-EphB2 or THEX1 antibodies are indicated in the upper part of the graph. All P values are calculated using Mann Whitney test by comparing Abs results from patients with SLE as a whole group or in SLE subgroups to all controls (CTLS).

Fig 4. Correlation between anti-THEX1 antibody titers and SLEDAI in patients with SLE.

Disease activity is indicated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) for 35 patients with SLE. Sera were tested at dilution 1/100 and defined as positive when Abs≥0 (on or above the dotted blue line). Spearman’s correlation r = 0.5843, P (two-tailed) = 0.0002.

Fig 5. ROC curve analysis with comparison of the ELISA for THEX1 and EphB2 in SSc and SLE.

Here are compared diagnostic abilities of EphB2 and THEX1 for SSc and SLE. Controls in both cases are all other individuals, which means patients with SSc were also included among controls when SLE is regarded as the tested disease and inversely patients with SLE were included among controls when SSc is regarded as the tested disease. Areas under the curve (AUC) for THEX1 and EphB2 in SLE are respectively 0,80 and 0,74. AUC for THEX1and EphB2 in SSc are respectively 0,59 and 0,58.

Fig 6. Autoantibodies against peptide 7 (P7 from EphB2 protein) analyzed in patients with scleroderma (SSc), lupus (SLE), Rheumatoid Arthritis (RA) and healthy controls (HC).

Sera reactivity against the peptide 7 (P7 at 10μg/well) is given by Absorbance (Abs) values for all patients and controls. Sera were tested at dilution 1/100 and defined as positive when Abs≥0 (on or above the dotted blue line). Red bars represent medians with interquartile ranges. Percentage of individuals positive for anti-P7 antibodies are indicated in the upper part of the graph. One data point for SLE is outside the Y axis limit (-0,41) and not represented here but counted for statistics. P value is calculated using Mann Whitney test by comparing patients with SLE to all controls (SSc, RA and HC, n = 157).

Fig 7. ROC curve analysis for peptide 7 from EphB2 protein (P7).

Here are compared diagnostic abilities of P7 for SSc and SLE. Controls in both cases are all other individuals, which means patients with SSc were included among controls when SLE is regarded as the tested disease and inversely patients with SLE were included among controls when SSc is regarded as the tested disease.

Fig 8. Correlation between anti-P7 antibody titers and SLEDAI in patients with SLE.

Disease activity is indicated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) for 48 patients with SLE. Sera were tested at dilution 1/100 and defined as positive when Abs≥0 (on or above the dotted blue line). Spearman’s correlation r = 0.3352, P (two-tailed) <0.02.