The detrimental effects of IFN-α on vasculogenesis in lupus are mediated by repression of IL-1 pathways: potential role in atherogenesis and renal vascular rarefaction

Abstract

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study, we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and β, IL-1R1, and vascular endothelial growth factor A, and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, by decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs, and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not anti-neutrophil cytoplasmic Ab-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1R antagonist, downregulation of vascular endothelial growth factor A, and glomerular and blood vessel decreased capillary density, compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.

Figure 1. Transcriptional network displaying IL-1β as a major node regulated by IFN-α on EPCs/CACs

Literature-based Genomatix Bibliosphere network was performed. The network generated from the regulated gene list between non-treated and IFNα-treated SLE PBMCs displayed IL-1β as one of the main nodes (1631 genes with a fold-change ≥ 0.15 and a q-value <0.05) (n=6 in each group). 1208 genes passed the Bibliosphere filter criterion, based on the co-citation of genes in PubMed abstract sentence linked by a function word (B2 filter). Only the 201 IL-1β directly connected regulated genes are displayed. The color code from red to blue indicates from up-to down regulation of genes in IFNα-treated SLE PBMCs compared to non-treated SLE PBMCs.

Figure 2. IL-1 family members and VEGF-A are regulated by IFN-α in lupus and control EPC/CACs

A. Regulation of IL-1β, IL-1R1, IL-1R2, IL-1RN and VEGF-A mRNA levels by IFN-α was confirmed by real-time PCR in EPCs/CACs from healthy controls and SLE patients. Results represent the mean ± SEM log fold change of IFN-α-treated over untreated cells. All treated samples were significantly altered with a p<0.05 when compared to untreated samples; (SLE n=11; control=17). B. SLE patients display elevated levels of IL-1RN in plasma (p=0.0116). Results represent mean± SEM plasma levels of IL-1RN in 35 SLE patients and 10 healthy controls.

Figure 3. IL-1β abrogates the abnormal phenotype of lupus EPC/CACs

A. Addition of recombinant IL-1β to lupus EPC/CAC cultures improves the ability of these cells to differentiate into mature endothelial cells to levels comparable to those of healthy controls (**p<0.01, n=24),while addition of recombinant VEGF-A resulted in a more modest but significant improvement (*p<0.05, n=24). Addition of IL-6 or TNF-α to the same cultures had no beneficial effect when compared to untreated cultures (n=9). Control cells showed no significant improvement in their capacity to differentiate into mature endothelial cells with any of the treatments mentioned above (n=7, n=5, n=3, n=3 respectively). Results are expressed as a ratio of the number of endothelial cells/ high power field to the number of untreated lupus endothelial cells/high power field; *p<0.05. B. Representative images displaying the effects of these proteins on EPC/CAC cultures from 2 SLE patients at 2-3 weeks. Images acquired at 100X total magnification. Bar indicates 200 um. Mature endothelial cells show coexpression of FITC-UEA-1(green) and uptake of DiI-Ac-LDL (red).

Figure 4. IL-1β increases proliferation and inhibits apoptosis of lupus EPC/CACs

A. Improvements in EPC/CAC proliferation by IL-1 and VEGF-A supplementation. Results represent mean ± SEM absorbance in duplicate wells; * p<0.05 (SLE: n=5 and controls: n=8). B. IL-1β inhibits activation of caspase 3 and 7 in lupus but not control EPC/CACs. Results represent mean fluorescent value of treated samples/mean fluorescent value of SLE media alone ± SEM; (SLE=5; control=8); * p<0.05.

Figure 5. IFN-α activates STAT2 and 6 on EPCs/CACs and JAK inhibition leads to downregulation of the antiangiogenic signature in SLE

Phosphorylation of STAT 2 and STAT6 was detected by FACS in EPCs and CACs. Stimulation with IFN-α leads to STAT2 and 6 activation in EPCs (A) and STAT2 activation in CACs. (B). Results represent mean % ±SEM of cells positive for STAT phosphorylation in 7 SLE and 4 control subjects. C. JAK inhibition induces upregulation of IL-1α, IL-1β, IL-1R1 and VEGF-A mRNA and decreases mRNA levels of IL-1RN and MX1 (D). Results represent average fold-change+SEM over vehicle-treated cells; SLE n=10 and control n=5; *p<0.05.

Figure 6. Kidneys from patients with lupus nephritis display decreased VEGF-A, increased IL-1RN and decreased glomerular and capillary density

A, and D. Representative photographs show that expression of IL-1RN is negative in the endothelium and vessel wall of a control kidney biopsy. In contrast, the vascular endothelium is clearly stained in a case of class V SLE nephritis and numerous positive cells are present in the vessel wall, particularly in the external layers. A mild vascular positivity for IL1RN can be detected in a medium sized vessel from a case of renal ANCA⁺ vasculitis (Immunoperoxidase, 400X). B and E. Evident loss of podocyte staining for VEGF-A in glomeruli from class IV lupus nephritis, as compared with control kidneys and ANCA⁺ vasculitis. (Immunoperoxidase, 200X). C and F. Compared to glomeruli from a control kidney and a case of vasculitis, glomerular expression of CD31 is significantly reduced in a case of SLE class III lupus nephritis. (Immunoperoxidase, 400X).