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Intronic single nucleotide polymorphisms (SNP) in STAT4, tagged by rs7574865[T], are associated with increased susceptibility to develop systemic lupus erythematosus (SLE) and a more severe disease phenotype with nephritis and stroke.1 2 Recently, we demonstrated that T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] have an enhanced induction of STAT4 protein following PHA/interleukin (IL)-2 activation, which results in increased IL-12-induced phosphorylation of STAT4 (pSTAT4) and interferon (IFN)-γ production.3 As the majority of STAT4 risk allele carriers do not develop disease, we asked whether the STAT4 risk allele exerts the same effect in healthy individuals. In studies of IL-12-stimulated cells from 72 healthy women (online supplementary materials and methods) we unexpectedly found that STAT4 risk allele carriership was associated with a decreased pSTAT4 in CD8+ and CD4+ T cells from healthy individuals (figure 1A). Notably, in the absence of STAT4 risk alleles healthy individuals and patients with SLE phosphorylated STAT4 to the same extent (figure 1B). However, with increasing numbers of STAT4 risk alleles, patients with SLE responded stronger to IL-12 stimulation. The decreased IL-12 response in healthy STAT4 risk allele carriers resulted in a decreased production of IFN-γ in CD8+ T cells and a slight, but non-significant, decrease in CD4+ T cells (figure 1C). Demonstrating the specificity for IL-12, phorbol 12-myristate 13-acetate-induced IFN-γ production was not affected (online supplementary figure 1). Thus, the STAT4 risk allele rs7574865[T] associates with opposite directional effects in cells from healthy donors compared with patients with SLE.
Opposing directional effects have been described for SNPs in terms of neurotransmitter receptor binding in patients with psychiatric diseases and healthy individuals,4 5 but to the best of our knowledge this is the first time such an effect is described in patients with SLE. Another example of this phenomenon is the differential regulation of mRNA expression in different cell types, and in in vitro stimulated cells compared with unstimulated cells.6 The underlying mechanisms for these observations are not completely known, but may include cell-type-specific expression or context-dependent induction of transcription factors.
When exploring these possibilities, we observed that basal mRNA levels of STAT4alpha, STAT4beta and STAT1alpha did not correlate with STAT4 genotype in healthy donor CD8+ memory T cells. However, following PHA/IL-2 stimulation, homozygous risk individuals displayed a defective induction of STAT4alpha mRNA expression, which is essential for IL-12-induced IFN-γ production,7 whereas STAT4beta and STAT1alpha mRNA were normally induced (figure 1D).
Given the important role of type I IFN in SLE, we hypothesised that IFN-α may be an environmental modulator of the STAT4 risk allele. Indeed, preactivation of healthy donor cells with IFN-α for 24 hours before PHA/IL-2 stimulation augmented the IL-12-induced pSTAT4 in CD8+ and CD4+ T cells from risk allele carriers, but not in non-risk allele carriers (figure 1E). In contrast, preactivation of cells with tumour necrosis factor-α did not affect IL-12-induced pSTAT4 (figure 1F). In support of an interaction with IFN-α and the STAT4 risk allele in patients with SLE in vivo, stratification of the patients from our previous study3 into those with plasma levels of IFN-α above or below the detection limit 0.5 U/mL revealed that the increased IL-12-induced pSTAT4 in CD8+ T cells from risk allele carriers was specific to the IFN-α+ patients (figure 1G).
In conclusion, the STAT4 risk allele rs7574865[T] has opposite effects in healthy individuals compared with patients with SLE and we identify IFN-α as an environmental modifier of the STAT4 risk allele. This finding may have implications for why the majority of risk allele carriers do not develop disease, and may also suggest a mechanism where STAT4 risk allele carriers are at risk to develop SLE when IFN is produced, for instance, during a viral infection.
We thank Martin Joelsson, Lisbeth Fuxler and Karolina Tandre for assisting in collecting the PBMCs. Genotyping was performed by the SNP&SEQ Technology Platform, which is part of Science for Life Laboratory and the National Genomics Infrastructure at Uppsala University, supported by the Swedish Research Council (VR-RFI) and the Knut and Alice Wallenberg Foundation.
Handling editor Josef S Smolen
Contributors NH performed the experiments and analysed the data. NH and LR designed the study, interpreted the data and wrote the manuscript.
Funding This work was supported by grants from the Swedish Research Council for Medicine and Health (D0283001), the Swedish Rheumatism Association, King Gustav V’s 80-Year Foundation, the Knut and Alice Wallenberg Foundation (2011.0073), the Swedish Society of Medicine and the Ingegerd Johansson donation, and Erik, Karin and Gösta Selander’s Foundation.
Competing interests None declared.
Patient consent Not required.
Ethics approval The study was approved by the Regional Ethics Board in Uppsala (2009/013).
Provenance and peer review Not commissioned; externally peer reviewed.
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