How the study of children with rheumatic diseases identified interferon-alpha and interleukin-1 as novel therapeutic targets

Abstract

Our studies in children with rheumatic diseases have led to the identification of two of the oldest cytokines, type I interferon (IFN) and interleukin 1 (IL-1), as important pathogenic players in systemic lupus erythematosus (SLE) and systemic onset juvenile arthritis (SoJIA), respectively. These findings were obtained by studying the transcriptional profiles of patient blood cells and by assessing the biological and transcriptional effect(s) of active patient sera on healthy blood cells. We also identified a signature that can be used to promptly diagnose SoJIA from other febrile conditions. Finally, our pilot clinical trials using IL-1 blockers have shown remarkable clinical benefits in SoJIA patients refractory to other medications.

Figures

Figure 1. Clinical features of pediatric SLE and SoJIA

A (top). Cutaneous involvement in a 12 year old girl with SLE; this patient also presented systemic manifestations including nephritis; (medium) light microscopy section showing lupus glomerulonephritis; (bottom) MRI showing lupus CNS involvement. B. (top) Salmon-colored rash typical of SoJIA in an 18 month old patient; (bottom) multiple joint swelling in the lower extremities of a 2 year old boy with SoJIA.

Figure 2. Dendritic cell alterations in SLE are driven by IFN-alpha

A. Monocytes from ¾ pediatric SLE patients induce significant proliferation of allogeneic naïve CD4+ T cells. B. SLE serum induces healthy monocytes to differentiate into cells with DC properties. C. An IFN-alpha neutralizing antibody blocks the induction of DCs by SLE serum, as measured using the proliferation of allogeneic T cells.

Figure 3. Fate of Autoreactive T Cells

Autoreactive thymic escapees (in red) are normally silenced in the periphery through the recognition of autoantigens presented by immature DCs (peripheral tolerance). An excess of IFN-αβ, as observed in SLE, induces unabated DC maturation, which leads to activation/expansion of autoreactive T cells. (Original figure published in Immunity 2004;20:539-50)

Figure 4. Plasmacytoid dendritic cells induce plasma cell differentiation through type I Interferon and Interleukin 6

Upon virus encounter, pDCs promptly secrete type I IFN. The cells differentiate into DCs presenting viral antigens to T cells, which promptly secrete IL-2 and turns on CD40-L that signals pDCs to secrete IL-6 and activates B cells. Activated B cells differentiate into plasma blasts in response to type I IFN. The secretion of IL-6 further induces the plasmablasts to become plasma cells. The T cells also contribute importantly through their secretion of IL-2 and IL-10, the combination of all signals leading to the generation of CD38++ long-lived plasma cells. (Original figure published in Immunity, 2003, 19: 225-234)

Figure 5. SLE signature

Hierarchical clustering of gene expression data by blood leukocytes of 9 healthy children, 30 with SLE and 12 with juvenile chronic arthritis including 3 systemic arthritis. The SLE patients were ranked according to their SLEDAI at time of blood draw. Each row represents a separate gene and each column a separate patient. 374 transcript sequences were selected as being differentially expressed in SLE by comparison to healthy patients. The normalized expression index for each transcript sequence (rows) in each sample (columns) is indicated by a color code. Red, yellow and blue squares indicate that expression of the gene is greater than, equal to or less than the mean level of expression across 9 healthy controls. The scale extends from fluorescence ratios of 0.25 to 4.0. (Original figure published in J. Exp. Med. 2003 Mar 17;197(6):711-23)

Figure 6. High dose steroid intravenous pulse extinguishes the type I IFN signature in SLE blood

Analysis of PBMCs from 3 pediatric SLE patients before and after treatment with high dose i.v. Methylprednisolone (1g/day for 3 days). All patients show down-regulation of IFN-regulated transcripts (upper panel) while expression of not type I IFN-inducible transcripts (lower panel) does not change significantly. p values on the right indicate significance of the gene expression level before and after steroid treatment (paired t-test). Original figure published in J. Exp. Med. 2003 Mar 17;197(6):711-23

Figure 7. A unified view of SLE pathogenesis

SLE flares are often associated to environmental triggers like viral infections. Thus, infection could trigger the unabated production of type I IFN in SLE patients. Increased bioavailability of type I IFN induces and maintains the generation of mature DCs, tilting the fate of autoreactive T lymphocytes which have escaped central tolerance from deletion to activation. These mature DCs activate cytotoxic CD8+ T cells to generate nucleosomes which can be captured and presented by IFN-DCs. Together with IL-6, type I IFN promotes the differentiation of mature B cells into plasma cells. Thus, the effects of type I IFN on DCs, B and T cells could explain the breakdown of tolerance to nuclear antigens, autoantibody secretion and IC formation characteristic of SLE. Innate immunity cells such as neutrophils may also contribute to lupus pathogenesis and end organ damage. (Original figure published in J. Exp. Med. 2003 Mar 17;197(6):711-23)

Figure 8. Gene expression profiling leads to the identification of IL-1 b in the pathogenesis of SoJIA

A) Analysis of transcriptional changes induced upon incubation of healthy PBMCs with autologous sera (AS) or with sera from four patients with active SoJIA (SYS33, SYS46, SYS53, and SYS27). Sera from patients induced the up-regulation of 46 genes. Median fold up-regulation by the four SoJIA sera incubation is depicted on the left column. The number of SoJIA sera that induced greater than twofold up-regulation is shown in the next column. B) Expression of a set of gene probes from Fig.1a in the PBMCs of 16 active SoJIA patients. The patient PBMCs expression data were normalized to the median expression of the same gene probes in the PBMCs of 12 healthy children. Median gene expression and number of samples with greater than twofold up-regulation are depicted in the first two columns. The third column represents the number of samples with a P (present) flag according to Affymetrix MAS 5.0 scaled gene expression data. p-values (Mann-Whitney test) are given next to these genes. (Published in Curr Opin Immunol. 2007 Dec;19(6):623-32)

Figure 9. Analysis of significance across diseases identifies 88 SoJIA-specific transcripts

(A) Eight healthy and eight SoJIA samples were used as training set to generate a list of 50 classifier genes displaying the best ability to discriminate SoJIA patients from healthy controls. Those classifier genes were hierarchically clustered in a test set composed of 35 healthy controls, 16 SoJIA, 31 S. aureus, 12 S. pneumoniae, 31 E. coli, 18 Influenza A and 38 SLE patients. (B) Genes expressed at statistically different levels in SoJIA patients compared to healthy volunteers (p<0.01, Wilcoxon-Mann-Whitney test) were selected (4311 probe sets). Out of those, 88 were found expressed at statistically different levels in SoJIA patients compared to healthy volunteers (p<0.01, Wilcoxon-Mann-Whitney test) but not in all the other groups (p>0.5, Wilcoxon-Mann-Whitney test). The 88 genes are hierarchically clustered in the 107 samples from different disease groups used in (A). Expression values or the genes are normalized per-gene to the healthy group (Published in Curr Opin Immunol. 2007 Dec;19(6):623-32)

Figure 10. Treatment with IL-1Ra (Anakinra) extinguishes the SoJIA-specific signature

(A). 88 SoJIA-specific genes were analyzed in 35 healthy, 22 SoJIA patients not receiving IL-1 blockers and 14 SoJIA patients after initiation of treatment with IL- blockers. *represents the same patients before and after initiation of the therapy. (B). The SoJIA signature is present in a patient on two occasions taken 2 years apart. On both occasions the patient was active and not receiving IL-1 blockers. (Published in Curr Opin Immunol. 2007 Dec;19(6):623-32)