We demonstrated that TLR9-induced IFN-α production is reduced in PBMCsPMBCs from SLE patients (Figure and Table ). SLE patients exhibit ongoing IFN-α production, and IFN-α serum levels are closely correlated with SLE disease activity [8
]. Although active SLE serum contains slightly increased levels of IFN-α as compared with inactive SLE and healthy control sera, there were no significant differences in serum levels between total or inactive SLE patients and healthy control individuals. Because some chronic SLE patients had lymphophenia (data not shown), the number of pDCs per blood unit was reduced in SLE patients, which would affect the serum levels of IFN-α. The proportion of circulating pDCs was slightly reduced in SLE patients, and IFN-α production was markedly impaired after in vitro
stimulation with TLR9, regardless of disease activity (Figures and ). Although SLE patients exhibited a slight, nonsignificant decrease in the proportion of pDCs to total PBMCs, we cannot exclude the possibility that this decrease contributes to the decrease in TLR-induced IFN-α production in SLE patients, because the composition of pDC subtypes may differ between SLE patients and healthy control individuals. There is evidence that the Ly6C/Ly49Q pDC subtypes are effective producers of IFN-α [33
], and so further investigation is required to determine the composition of pDC subtypes in SLE patients. Other studies have reported that SLE patients exhibit a reduced number of BDCA-2 expressing pDCs, and that herpes virus induced IFN-α production is decreased in SLE PBMCs [16
]; furthermore, CpG-induced IFN-α secretion was significantly reduced in monocytes and dendritic cells from SLE patients [18
]. However, CpG-induced IFN-α production was completely abolished in one-third of SLE patients, and the decrease in IFN-α production was more marked than the decrease in pDCs, indicating that a different mechanism is at play.
SLE patients exhibited decreased numbers of circulating pDCs (Figure ), which is consistent with the findings of a number of other studies [16
], but they also showed increased numbers of pDCs in cutaneous lesions [35
]. It has been suggested that circulating pDCs are low in SLE patients because this cell type is recruited from the blood to peripheral tissues. However, the fate of circulating pDCs after activation by DNA-containing immune complexes, which present in the blood of SLE patients, is not yet clear. Our results showed that significant numbers of pDCs are still present in the PBMC fraction isolated from SLE patients. Furthermore, we demonstrated that TLR-tolerant pDCs can recover over time and restore IFN-α production (Figure ), suggesting that pDCs in SLE patients are still present but inactive as a result of TLR tolerance or exhaustion.
The marked decrease or abrogation of IFN-α production may be explained by factors other than cell count. We noted that CpG-induced IFN-α production in SLE PBMCs was inversely correlated with SLE serum-stimulated cytokine production in healthy PBMCs (Figure ). We also found that repeated or chronic stimulation of TLR9 by appropriate ligands, such as CpG ODN 2216 or DNA-containing immune complexes, leads to TLR tolerance in pDCs. Although the mechanism of TLR tolerance has not been fully explained, it is a well known occurrence for cells that have been persistently stimulated with TLR ligands to fail to respond to re-stimulation [31
]. One possible mechanism is inhibition of TLR signaling via dysregulation of lipopolysaccharide-induced TLR4-MyD88 complex formation and IL-1 receptor-associated kinase (IRAK)-1 activation in endotoxin-tolerant cells [38
]. Another possibility is induction of genes that negatively regulate TLR signaling, such as IRAK-M and suppressor of cytokine signaling (SOCS)-1 [31
We found an increase in the expression of IFN-α signature genes, indicating that SLE PBMCs have already been exposed to IFN-α, which is mainly produced by pDCs (Figure ). Although we did not check the expression levels of molecules that inhibit the TLR signaling cascade in pDCs from SLE patients, the SLE PBMCs showed elevated levels of IRAK-M and MyD88s compared with the healthy PBMCs (Additional file 1
[Supplementary Figure 3a,b]). Because inflammation may also increase the expression of TLR signaling molecules, we examined the expression of MyD88, which is a positive regulator of the TLR signaling pathway. MyD88 expression was also slightly elevated in SLE PBMCs (Additional file 1
[Supplementary Figure 3c]), although the ratio of MyD88s to MyD88 indicated that the negative regulator, MyD88s, was dominantly expressed in the SLE PBMCs (Additional file 1
[Supplementary Figure 3d]). Although these data do not reveal the functional status of the pDCs in SLE patients, they suggest that the expression of negative regulators of TLR signaling may be responsible for the development of TLR tolerance in the PBMCs of SLE patients. In addition, dysfunctional IFN-α production by SLE pDCs can be induced by other TLR ligands that are found frequently in SLE sera, such as RNA-containing immune complexes and heat shock proteins. However, our investigation was hampered by the limited number of pDCs that could be isolated from the available blood sample, and thus the exact mechanism of TLR-9 tolerance remains to be elucidated. Further investigation is required to clarify this issue.
Another possible mechanism for TLR tolerance is that SLE medications may affect the function of pDCs. Although no correlations were observed among serum IFN-α levels, CpG-induced IFN-α production in vitro
, and the type and dosage of medicines taken by SLE patients (data not shown), the immuno-suppressors, such as cyclosporine and hydroxychloroquine, can affect the function of pDCs. Hydroxychloroquine, in particular, is a known inhibitor of TLR9 signaling; this drug blocks the acidification of endosomes (phagosomes), which is essential for TLR9 signaling [40
]. To rule out the effect of hydroxychloroquine, pDCs from healthy individuals were pretreated with 1 mmol/l hydroxychloroquine for 24 hours, washed twice in serum-free medium, and then treated with CpG ODN2216. After 24 hours of incubation, IFN-α production decreased by up to 60% compared with non-pretreated pDCs (data not shown). These findings indicate that the residual amounts of hydroxychloroquine in pDCs from SLE patients may contribute to TLR tolerance. Moreover, we cannot exclude the potential influences of other medications on pDC numbers and functions. However, not all SLE patients were taking hydroxychloroquine, and the inhibition of TLR9 by residual hydroxychloroquine cannot fully explain the abrogation of IFN-α observed in one-third of SLE patients (Figure ).