Background
Systemic lupus erythematosus (SLE) is characterized by circulating immune complexes (ICs), an activation of the type I interferon (IFN) system, and production of proinflammatory cytokines and chemokines which cause an autoimmune reaction with organ inflammation [
1]. The cellular and molecular mechanisms behind the ongoing inflammatory process in SLE have been partially clarified, and a number of different disease-associated pathways identified [
2]. One important event is the induction of type I IFN production by plasmacytoid dendritic cells (pDCs) in response to ICs consisting of autoantibodies and apoptotic or necrotic cell-derived nucleic acids [
3]. Such interferogenic ICs are internalized in pDCs via fragment crystallizable receptor IIA (FcγRIIA) and directed to the endosomes, where RNA and DNA interact with Toll-like receptor (TLR)7 and 9, respectively [
4]. Activation of TLR7/9 triggers a signaling cascade, involving myeloid differentiation primary response protein 88 (MyD88), interleukin (IL)-1 receptor-associated kinase (IRAK)1, and IRAK4, that eventually leads to transcription of type I IFN genes. In addition to type I IFN production, MyD88 and IRAK4 signaling triggers the production of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-6, via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) or IFN regulatory factor (IRF) 5 [
5]. Besides the pDCs, interferogenic ICs will also activate several other immune cells, such as natural killer (NK) cells, which contribute to enhanced cytokine production [
6]. The final outcome in SLE is a complex inflammatory response that is difficult to bring into complete remission.
Current therapies in SLE aim to downregulate the autoimmune reaction. Treatment with antimalarials, such as hydroxychloroquine (HCQ), is considered the standard of care [
7,
8]. The presumed central mechanism of action of HCQ is a reduction in the IFN-α production by inhibition of endosomal TLR signaling [
9]. Studies have also shown that SLE patients treated with HCQ have a decreased type I IFN production after stimulation of pDCs with TLR ligands [
10]. However, despite continuous HCQ treatment, few patients with SLE experience complete remission and flares still occur. A possible reason could be the limited number of disease-associated pathways affected by HCQ. Consequently, targeting a broader repertoire of inflammatory cytokines in SLE, yet avoiding severe infections, is needed. A potential therapeutic target in SLE is IRAK4 due to its essential role in MyD88 signaling [
11]. IRAK4-deficient children are susceptible to life-threatening pyogenic infections that are reported to cease in adolescence, making IRAK4 inhibition an attractive therapeutic possibility [
12].
In this study, we compared the effect of HCQ and the IRAK4 inhibitor (IRAK4i) I92 on the RNA-IC-induced cytokine production by pDCs and NK cells from healthy individuals and monocyte-depleted peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls. Gene expression profiles of RNA-IC-stimulated pDCs treated with IRAK4i or HCQ were compared with nontreated cells to clarify the inflammatory response modulated by the drugs.
Discussion
This study demonstrates that the cytokine production by RNA-IC-stimulated pDCs and NK cells can be suppressed by HCQ and, more profoundly, by an IRAK4 inhibitor. The strong TNF-α induction by RNA-IC is interesting since TNF-α plays a critical role in several SLE disease manifestations, such as nephritis, skin lesions, and arthritis, all characterized by tissue deposition of ICs [
26‐
28]. Increased IC formation precedes SLE flares and our findings may therefore partly explain the observed association between increased serum TNF-α levels and disease activity in SLE [
29]. The difference in the TNF-α production rate between pDCs and NK cells indicates that RNA-ICs activate different induction pathways for TNF-α synthesis in these two cell types. Supporting this conjecture is the observed difference between pDCs and NK cells in response to HCQ. pDCs are mainly activated by ligation of endosomal TLRs [
2] and this pathway is inhibited by HCQ [
7]. TNF-α production by NK cells, on the other hand, can be induced by a number of different receptors, including TLR7 [
30‐
32]. However, RNA-IC-induced production of cytokines and chemokines from NK cells was not dependent on endosomal TLR signaling since HCQ had no inhibitory effect. Consistent with a TLR7-independent activation of NK cells, heat-aggregated IgG was as efficient as RNA-IC in inducing TNF-α from purified NK cells, and a synthetic TLR7 agonist (DSR6434) did not induce TNF-α in NK cells (Additional file
13). Although no studies were performed regarding specific RNA-IC-responding NK cell receptors, the fact that TNF-α production was observed only in the CD56
dim, CD16-expressing NK cell population (Additional file
14) suggests NK cell activation by RNA-IC via CD16. The prominent effect of the IRAK4 inhibitor I92 on the TNF-α production by NK cells implies that NFκB-mediated and/or mitogen-activated protein kinase activation was involved in the NK cell response [
33]. However, we cannot exclude that other protein kinases were also affected by I92, despite the previously demonstrated high selectivity for IRAK4 by this drug [
16].
When investigating the effect of HCQ and IRAK4 inhibitor I92 on other RNA-IC-induced cytokines, we noted that HCQ almost completely blocked the production of all investigated cytokines by pDCs. This was in stark contrast to the lack of effect on the cytokine response in NK cells. Conversely, HCQ markedly reduced the production of most cytokines in the pDC/NK cell cocultures. The reason for this is unclear, but an optimal cytokine production in cell cocultures depends on both cell types since RNA-IC-activated pDC and NK cells promote the function of each other [
6]. Consequently, inhibition of the pDC function in pDC/NK cell cocultures will also reduce the NK cell cytokine-producing capacity. However, in pDC/NK cocultures the production of IFN-γ and MIP-1β was not affected by HCQ, suggesting a pDC-independent production by NK cells, although the exact cellular source of these cytokines was not investigated. Nevertheless, this observation indicates the need for a therapeutic agent with broader effects than HCQ to achieve better control of IC-driven inflammatory processes.
The IRAK4 inhibitor I92 blocked the NK cell production of all cytokines in healthy individuals, except for IL-8. This could imply yet another induction mechanism for IL-8 production in NK cells. In fact, IL-8 production was also remarkably high in monocyte-depleted PBMC cultures from SLE patients (Additional file
15) but, due to a shortage of patient material, the effects of I92 and HCQ on the IL-8 production could not be clarified. Studies have shown that patients with SLE have increased serum levels of IL-8 despite continuous standard treatment and being in remission [
34]. An association between IL-8 gene polymorphisms and SLE further supports a role for IL-8 in SLE [
35]. Additional studies are needed to determine the regulation of IL-8 production in patients with SLE, and some are now in progress. Notably, I92 inhibited all other investigated cytokines produced by RNA-IC-stimulated cells from SLE patients, whereas HCQ only reduced IL-6 and MIP1-β production significantly.
The RNA-IC activation signature in pDCs revealed an enrichment of pathways with connection to the IFN signaling system, antigen presentation, and apoptosis. This demonstrates that nucleic acid containing ICs elicit a powerful inflammatory response, but also trigger other cellular processes of importance in SLE. Both I92 and HCQ largely reversed the RNA-IC activation in pDCs, although some differences were observed. I92 increased the expression of genes involved in protein degradation and the autophagy process, in contrast to HCQ which downregulated these genes. The most strongly downregulated genes by I92 compared with HCQ were
DKK4,
LAD1, and
EAF2, and suppression of these genes could have several effects on the SLE disease process. DKK4 is an inhibitor of the canonical Wnt signaling pathway, which has been suggested to contribute to disrupted T effector cell differentiation and the immune dysfunction in SLE [
36,
37]. Ladinin 1, encoded by
LAD1, modulates the EGF to ERK pathway and increased ERK activation is associated with organ damage in SLE [
24,
38]. EAF2, on the other hand, is selectively upregulated in germinal center B cells and promotes their apoptosis [
39]. Possibly, inhibition of EAF2 could therefore increase autoantibody production. The increased activation of the autophagy pathway by I92 might be beneficial in SLE since autophagy is reduced in SLE regulatory T cells and enhanced autophagy has been shown to improve both murine and human SLE [
40]. On the other hand, activation of autophagy favors plasmablast development, enabling expansion of self-reactive B cells in SLE, as well as type I IFN production by facilitating intracellular IC transport [
41,
42]. These observations merit further studies of the effects of I92 on different cell types, not least considering that IRAK4 inhibition ameliorates experimental murine lupus, suggesting a favorable effect also in human SLE [
43]. Translating results of in-vitro studies of pharmaceutical compounds to potential drug effects in vivo has limitations. However, the approach to investigate drug candidates in cell cultures can be useful to determine the effects on central immune cells in the disease process [
44]. Thus, we consider our system with IC-stimulated immune cells from SLE patients as one relevant model for an initial screening of potential drugs that target disease-associated pathways in SLE.
Acknowledgements
We thank Lisbeth Fuxler for excellent technical assistance, Rezvan Kiani Dehkordi for collecting the patient blood samples, and Dr. Gert Weber, Ernst-Moritz-Arndt University of Greifswald, for kindly providing the U1snRNP particles.