The present study shows that IKK-neutralizing compound BAY11 affects IFN-α production mainly through its action on pDCs. IFN-α production is differentially regulated from other inflammatory cytokine production by the specific intracellular signaling under TLR activation [40
]. A key molecular switch responsible for IFN-α synthesis in pDCs is the nuclear translocation of IRF7 [5
]. We here found that BAY11 inhibits the nuclear translocation of IRF7 in pDCs and their IFN-α production. Although there are a number of reports showing the potential use of BAY11 in the treatment of malignancies through its inhibitory activity of NF-κB, the evidence linking it to autoimmune diseases is scant and there is no direct evidence so far that BAY11 prevents the activity of type I IFN-related diseases such as SLE. pDC activation in the blood by self-nucleic acids is regarded as a pathogenic trigger of the autoimmune process, and a dysregulated type I IFN elevation in serum by the continuous pDCs activation amplifies the pathogenic spiral in SLE [12
]. On the basis of our current results showing that BAY11 inhibited the IFN-α production in PBMCs from SLE patients as well as from healthy donors, treatment with BAY11 may have the potential to attenuate the IFN environment and in turn to break off the pathogenic spiral in autoimmune diseases by limiting the disordered pDC function. Also, the experiments in injecting mice with poly U are suggestive of the agent's potential in inhibiting the inducible IFN response in vivo
, though the serum IFN elevation is not pathophysiologically but artificially induced in our experimental setting.
Under normal physiological conditions, host-derived self-nucleic acids usually have little chance of encountering endosomal TLR7 and TLR9 because of their instability in relation to nucleases and by their location separate from endosomes. However, a breakdown in the innate tolerance to self-nucleic acids occurs when tissue injury or necrosis release some endogenous molecules, including antimicrobial peptide (LL37) and nuclear protein (high-mobility group box 1 protein; HMGB1), which help to promote stabilization and delivery of immune complexes into early endosomes [9
]. Even in the current experiments using SLE sera and necrotic cell supernatant that perhaps comprise these molecules, BAY11 functions as an inhibitor of the pathogenic IFN-α response. Thus, our findings provide an opportunity for the development of therapeutic strategies that directly inhibit the pathogenic cellular and molecular components leading to SLE.
Also TNF-α production in pDCs was repressed by BAY11 at the high concentration, and accordingly the therapeutic window of BAY11 for selective interference with IFN-α was narrow. Since endogenous TNF-α limits the IFN-α production in pDCs [43
], there is a possibility that the repression of TNF-α results in abating the inhibitory function of BAY11 against IFN-α production at high concentration. Thus, the most efficient and practical biological concentration may need to be decided from further studies.
At the downstream of TLR7/9-MyD88, the signaling pathway bifurcates into NF-κB- and IRF-7- activation pathways, which are responsible for the induction of proinflammatory cytokines and type I IFNs, respectively [2
]. Whereas IRF7 phosphorylation and nuclear translocation depend on IKKα, NF-κB activation needs IKKβ. IKKβ homodimer can compensate the function of heterodimer of IKKα and IKKβ in activating NF-κB in the absence of IKKα [40
]. Given the function of BAY11 as an inhibitor of IKK activity [18
], a more plausible explanation for its inhibitory activities in regards to both IFN-α and TNF-α in pDCs is that BAY11 targets IKKα in the inhibition of IFN-α and IKKβ in the inhibition of TNF-α at the downstream of TLR7/9-MyD88.
The other two IKK-related kinases, TANK-binding kinase 1 (TBK1) and IKKτ (also called as IKKε), are also reported to be involved in the phosphorylation of IRF-7 as well as IRF3 [44
]. However, CpG-induced IFN-α secretion is not impaired in mice deficient in TBK1 or IKKτ [7
], indicating that these two IKKs are dispensable for TLR-mediated induction of IFN-α in pDCs. Similar to IKKα deficiency, IRAK1 deficiency leads to the defective transcriptional activation of IRF7 and defective production of IFN-α gene in pDCs [8
], indicating a critical involvement of IRAK1 in the induction of type I IFNs in TLR7 and TLR9 signaling pathways. Although it is unclear at present how IKKα links to IRAK1, either kinase appears to be the gateway for activation of IRF7 to induce IFN-α production in pDCs and both could be potential targets for the treatment of autoimmune disorders. Further studies will be required to determine what the specific target of BAY11 is, whether BAY11 inhibits IRAK1 activationor the precise mechanism by which BAY11 inhibits the signaling pathway of TLR-mediated IFN-α production in pDCs.
In contrast to RNA-sensing receptor TLR7 in pDCs, another RNA-sensing cytosolic RIG-I-like receptor sensors in myeloid DCs through recognition of dsRNA such as poly IC can also induce IFN-α/β in an IPS-1-dependent manner [45
]. However, BAY11 was incapable of inhibiting the poly IC-induced IFN-α production by myeloid DCs. This finding can be explained by the evidence that, at the downstream of RIG/MDA5-IPS-1, both IKKα and IKKβ are dispensable for the type I IFN production [9
]. However, BAY11 could suppress the poly IC-induced IL-12 and TNF-α secretion by the myeloid DCs. This could also be explained by the evidence showing that TLR3-mediated production of proinflammatory cytokines is dependent on IKKβ during the signaling process of the TRIF-NF-κB pathway [40