Plasmacytoid DC are thought to be the principal source of IFN-α production in most situations (17
). However, cDC are also able to produce appreciable levels of IFN-α under certain conditions such as with viral infection by cDC-tropic dsRNA viruses (22
) or after internalization of class A CpG oligodeoxynucleotides (ODN) by transfection with liposomes (23
). In this study we show that TLR4 activation by LPS is able to induce robust IFN-α production by mouse cDC and human monocytes that have been pretreated with type I IFN.
The critical role of the type I IFN feedback loop as an enhancer of IFN-α production is well established (55
). This refers to be ability of small amounts of type I IFN to engage the type I IFN receptor and thereby induce a variety of receptors, adaptors, and transcription factors which serve to augment IFN-α production in response to a subsequent stimulus. While this has been studied most extensively in fibroblasts, it also plays an important role in other cell types including DCs, although pDC may be less reliant on this feedback loop under some circumstances (57
). It has therefore been puzzling why although TLR4 is able to strongly induce IFN-β gene expression it does not generally induce IFN-α gene expression in vitro (other than IFN-α4) (18
). This contrasts with TLR3 which is able to induce both IFN-β and IFN-α (19
). Even with IFN-β pretreatment of GM-CSF-derived cDC for 16 h, a previous study found no induction of IFN-α gene expression with LPS activation (18
). However, in human macrophages pretreated with IFN-α for 16 h, LPS did induce IFN-α gene expression (58
). Our findings provide one explanation for this apparent discrepancy by showing that the length of pre-treatment with IFN-β is critical, with the priming effect being maximal with 5 h of pre-treatment and declining thereafter. Thus, at least in cDC, a pretreatment duration of 16 h may be too long to sustain the priming effect. This could potentially be due in part to the short-half life of certain of the components of the IFN-α inducing pathway that are upregulated by IFN-β pre-treatment, for example IRF7 which has a half-life of about 1 h (54
However, the TLR9 ligand CpG-A was unable to induce IFN-α in cDC, despite the established ability of type I IFN pre-treatment to upregulate many components of the TLR9 pathway required for IFN-α production in pDC such as MyD88, and IRF7 (18
). Furthermore, CpG-A was able to induce IL-6 in cDC demonstrating intact TLR9 signaling pathways and a selective impairment of IFN-α induction. This indicated that additional factor(s) specific to the TLR4 signaling pathway must be induced by IFN-β pretreatment that enabled IFN-α production following TLR4 activation.
IL-6 likely plays a critical role in the B cell hyperactivity and immunopathology of human SLE (61
), and has been linked to disease pathogenesis in murine lupus (62
). IL-6 and type I IFN also mediate protective effects against a variety of infectious organisms (63
). In this study we found that DC supernatants containing type I IFN (lupus IgG sup 1 and 2, and ), but not DC supernatants containing IL-6 (Pam3Cys sup, and ), could prime cDC for TLR4-induced IFN-α production. We have previously reported that IL-6 production by DC in response to a number of different TLR ligands including TLR4 ligands is type I IFN-dependent (40
). Thus although IL-6 does not directly prime cDC for type I IFN production, in the context of an infection, the type I IFN induced by TLR4 activation could lead to enhanced IL-6 production through this feedback loop. The increased levels of IL-6 and type I IFN could then contribute to recovery from the infection but worsening of the SLE.
Most TLRs are absolutely dependent on the expression of MyD88 for signaling. However, TLR3 signaling is completely MyD88-independent and TLR4 signaling partially MyD88-independent with both TLR3 and TLR4 being unique in their ability to utilize the adaptor TRIF (26
). TRIF expression is up-regulated by type I IFN pretreatment (58
), and this therefore represents one possible mechanism whereby IFN-β pre-treatment might enable IFN-α production by cDC in response to LPS. Consistent with this possibility, LPS failed to induce IFN-α production in TRIF-deficient cDC.
Unexpectedly however, we found that IFN-α and IFN-β protein production induced by LPS in type I IFN-pretreated cDC was also substantially MyD88-dependent. This contrasted with IFN-α and IFN-β induced by poly I:C which we found to be entirely MyD88-independent consistent with the findings of other investigators (30
). Most studies evaluating IFN-β production in response to LPS have focused on gene expression rather than protein production and have examined events soon after LPS stimulation. These studies clearly demonstrate that the early induction of IFN-β gene expression after LPS stimulation is MyD88-independent (20
). However, where later time points have been examined, lower levels of IFN-β gene expression were found in MyD88-deficient DCs compared with MyD88-sufficient DCs (20
). Our findings with IFN-β protein production demonstrate that early production is MyD88-independent (but TRIF-dependent) whereas later production is largely MyD88-dependent. IFN-α protein production from IFN-β pretreated cDC induced by LPS followed a very similar course. The TLR4-mediated induction of pro-inflammatory cytokine genes such as TNF and IL-6 also depends on both the MyD88 and TRIF pathways (26
). However, in the case of pro-inflammatory cytokines, MyD88-deficient cells lack early activation whereas TRIF-deficient cells show impairment in late activation (66
), the opposite of what we found for IFN-α and IFN-β production. This concept of co-operative interaction between the TRIF and MyD88 pathways is further supported by a number of studies showing synergy between ligands for TRIF-associated TLRs and ligands for MyD88-associated TLRs for the production of inflammatory cytokines (48
Previous studies have shown that IRF3, rather than IRF7, is essential for LPS-induced IFN-β gene induction because induction of the IFN-β gene in response to LPS is abolished in DCs from IRF3-deficient DCs (18
), but is almost normal in DCs from IRF7-deficient mice (35
). However, when DCs are pretreated with recombinant IFN-β, LPS is able to induce IFN-β mRNA expression in DCs from IRF3-deficient mice (18
). This suggests the possibility that IRF7, which is strongly upregulated in DCs by IFN-β, might participate in TLR4 signaling under certain conditions (18
). Our results demonstrate that IRF7 is absolutely required for LPS-induced IFN-α production by IFN-β pretreated DCs. This formally establishes that IRF7 can participate in TLR4 signaling and is consistent with the established role of IRF7 as the master regulator of IFN-α responses (36
The exact role of IRF5 in type I IFN production is less well established. IRF5 was originally identified in cell lines as a regulator of type I IFN gene expression induced by infection with certain viruses (37
), and is a central mediator of type I IFN production induced following TLR7 activation by R848 in HEK293 cells (39
). In vivo, IRF5-deficient mice are vulnerable to viral infections, and have a reduced level of type I IFN in their sera (41
). IRF5 also plays an important role in pro-inflammatory cytokine production (45
). We have previously found that IRF5 participates in the production of both IFN-α and IFN-β induced by TLR3, TLR7, and TLR9 ligands in DCs in vitro (40
). In this study, we confirm the involvement of IRF5 in IFN-α production induced by TLR3 and TLR9 ligands, and further show that IRF5 also participates in IFN-α production induced by TLR4 signaling.
Monocytes have the capacity to differentiate into either tissue macrophages or conventional DCs. They comprise up to 10% of circulating PBMC in humans, express high levels of TLR4, and respond to LPS (52
). We found that human monocytes pretreated with IFN-β behaved similarly to mouse cDC in terms of their ability to produce IFN-α on LPS stimulation. Although the amount of IFN-α produced per cell was relatively modest, it is possible that this could translate into substantial levels in vivo in the context of bacterial infection given the large numbers of monocytes present in the circulation. Thus, in situations where monocytes may have been exposed to type I IFN such as in SLE, bacterial infection could further increase IFN-α levels and initiate or exacerbate clinical disease. Cell types other than monocytes could also potentially play a similar role. For example, IFN-α pretreated macrophages express type I IFN following TLR4 triggering (58
). Human myeloid DC and plasmacytoid DC express little or no TLR4 and so are unlikely to contribute substantially to LPS-induced IFN-α production (52
Studies of hospitalized patients have shown that overall disease activity measured by either the SLE Disease Activity Index (SLEDAI) or the Lupus Activity Index (LAI), correlates well with the incidence of infection, the majority of which are bacterial (11
). In mouse models of lupus, administration of LPS greatly accelerates disease (71
). While these studies do not directly implicate type I IFN in pathogenesis, it is noteworthy that many of the toxic effects of LPS in vivo require type I IFN induction (72
), and type I IFN induces sensitization to subsequent LPS challenge (73
). Endogenous TLR4 ligands could act similarly. In this context, modified low-density lipoproteins and fatty acids have been identified as TLR4 ligands (75
) and could potentially contribute to the worsening of lupus disease activity seen in the setting of elevated low-density lipoprotein levels (78
). TLR4 transgenic mice expressing multiple copies of TLR4 develop lupus-like autoimmune disease in the absence of exogenous stimuli suggesting that either endogenous TLR4 ligands or commensal bacteria contribute to disease pathogenesis in this setting (79
). In summary, we describe a novel pathway for TLR4-induced IFN-α production by cDCs and suggest a possible mechanism whereby bacterial infection or endogenous TLR4 ligands could precipitate disease flares in SLE.