The tolerogenic peptide, hCDR1, down-regulates the expression of interferon-α in murine and human systemic lupus erythematosus.

The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus.(NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1β, TGFβ and FoxP3 gene expression.We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus

hCDR1 down regulates IFN-α gene expression in PBMC of SLE patients.

<p>PBMC of 10 SLE, 5 primary APS patients and 5 healthy controls were cultured (5×10⁶ cells/well) for 48 hours in the presence of medium or hCDR1 (25 µg/ml). Gene expression was determined by real-time RT-PCR. Results are presented as the mean±SE percentage of gene expression compared with cultures with medium (considered as 100%). * p = 0.004.</p

IFN-α diminishes hCDR1 immunomodulatory effects on PBMC of SLE patients.

<p>PBMC of 3 SLE patients were cultured (5×10⁶ cells/well) for 48 hours in the presence of medium, hCDR1 (25 µg/ml) or hCDR1 (25 µg/ml) and human recombinant IFN-α (rIFN-α) at a concentration of 5,000 U/ml. Gene expression (for IL-1β, TGFβ and FoxP3) were determined by real-time RT-PCR. Results are presented as the mean±SEpercentage of gene expression compared with cultures of PBMC incubated with medium alone (considered as 100%). *p = 0.05, **p = 0.03, ***p = 0.015 and ⁰ = not significant.</p

Effects of treatment with hCDR1 on SLE manifestations in mice.

a<p>SLE-afflicted (NZB×NZW)F1 mice (10–12 mice per group in 4 independent experiments) were treated with weekly subcutaneous injections of the vehicle, hCDR1, or a control (scrambled peptide) for 10 weeks.</p>b<p>Results are of sera from mice that were bled after the end of treatment. Dilution of sera 1∶1250.</p>c<p>Statistical evaluation was based on the Mann-Whitney U test to compare post –treatment effects between the vehicle –treated groups and the remaining treatment groups.</p>d<p>Proteinuria was always measured at about the same time of day and all mice in an experimental cohort were tested together.</p>e<p>Immune complex deposits (ICD) were assessed at sacrifice.</p>f<p>p = 0.04, 0.02 and 0.0001 between the control peptide and hCDR1 treated mice for dsDNA specific antibodies, proteinuria and ICD, respectively.</p

Treatment of SLE afflicted (NZBxNZW)F1 mice with hCDR1 results in the down regulation of IFN-α.

<p>(A) Mean percentage (±SE) results of 4 independent experiments in which the mRNA expression of IFN-α was determined in pools of spleen derived cells of SLE afflicted mice (10–12 mice per group) treated with vehicle, hCDR1, or the control, scrambled, peptide. The levels of gene expression were determined by real-time RT-PCR and calculated relatively to levels in cells from vehicle-treated mice (considered as 100%). (B) Mean concentrations (±SE) of IFN-α determined by ELISA in sera of the same groups of mice.</p