Recent studies strongly suggest a link between elevated IFN-I production and the pathogenesis of SLE. More than half of SLE patients display increased expression of ISGs, often in association with active disease and autoantibodies against snRNPs and DNA, as well as renal involvement and endothelial dysfunction (2
). However, the exact cause of IFN-I dysregulation in lupus remains controversial, and it is unclear whether IFN-I overproduction promotes autoantibody production or vice versa.
The TMPD model of lupus recapitulates many features of human SLE, including glomerulonephritis, arthritis, and autoantibodies against dsDNA and snRNPs (25
). Moreover, like SLE, TMPD-induced lupus is more severe in females than males (46
). We recently found that TMPD-treated mice exhibit the IFN signature and that their lupus is dependent on IFN-I signaling (22
). The major IFN-I–producing cells in TMPD lupus are Ly6Chi
monocytes rather than PDCs (27
). In this study, we show that TMPD triggers IFN-I production via the TLR7–MyD88 pathway. Our data also exclude a major role of other pathways of IFN-I production, including TLR9, TLR3–TLR4–TRIF, RIG-I–Mda5–IPS-1, and DAI–TBK1. Although there was a strict requirement for TLR7 and MyD88, expression of ISGs and recruitment of Ly6Chi
monocytes in response to TMPD was unexpectedly independent of FcγRs.
The innate sensors TLR7 and TLR9 have been implicated in SLE because of their ability to recognize endogenous nucleic acids and trigger IFN-I production (47
). Mammalian nucleic acids are generally weak TLR ligands because of their inability to reach the endosomal compartment where TLR7 and TLR9 are localized. When they form ICs with lupus autoantibodies (anti-Sm/RNP or anti-dsDNA), endogenous nucleic acids may be delivered more efficiently to endosomes because of uptake by FcγRs, stimulating IFN-I production by PDCs (17
). This model for IFN-I induction in human SLE is supported by numerous in vitro studies (19
). Although FcγRIIa mediates the activation of human PDCs, ICs trigger IFN-I production by mouse PDCs in a TLR7- and FcγRI/III-dependent manner (33
However, it is not known whether the development of antiribonucleoprotein autoantibodies is a cause of IFN-I dysregulation or a consequence of it. Therapeutic use of IFN-α can induce many features of SLE, including anti-dsDNA antibodies, suggesting that IFN-I dysregulation occurs upstream of IC formation. Consistent with that view, IFN-I up-regulation in the TMPD model occurs within the first 2 wk of treatment, more than 2 mo before the onset of lupus autoantibodies (27
). FcγRI and FcγRIII were not required for the IFN-I response to TMPD because it was unaffected in γ chain–deficient mice. Moreover, a previous study also showed that the absence of FcγRI/III or FcγRIIb does not affect anti-Sm/RNP autoantibody production in TMPD-treated mice (49
). Although IC formation and FcγRs are not required to initiate IFN-I production, we cannot exclude the possibility that they amplify IFN-I secretion and accelerate disease progression subsequent to the development of autoantibodies.
A pathogenic role of TLR7 has been described in several mouse models of SLE. In MRL-lpr
mice, TLR7 ligands accelerate the onset of glomerulonephritis, whereas deletion of TLR7 abrogates the development of anti-Sm autoantibodies and lessens the severity of kidney disease (41
). Lupus in MRL-lpr
mice has been reported to be ameliorated, not exacerbated, by IFN-I (51
), and the lpr
defect prevents induction of TMPD lupus (52
Dual engagement of TLR7 and the B cell receptor can directly activate autoreactive B cells in the AM14 model, and TLR7 is also required for the spontaneous production of autoantibodies against ssRNA in 564Igi transgenic mice (53
). The connection between TLR7 and the generation of RNA-associated autoantibodies is further illustrated by the recent demonstration of a duplication of the TLR7 gene in Yaa mice. The presence of the Yaa cluster was sufficient to induce production of RNA-associated autoantibodies in C57BL/6 FcγIIB−/−
and C57BL6.Sle1 mice, two autoimmune strains that normally lack these antibody specificities (38
). TLR7, and not the other 16 genes affected by the Yaa mutation, is responsible for the autoimmune pathology (40
). However, increased IFN-I production has not been reported in these models. Our findings indicate that TLR7 also plays an essential role in TMPD lupus. Similar to the MRL-lpr
), TLR7 is required for the generation of anti-nRNP/Sm autoantibodies in TMPD-treated mice. Importantly, the IFN signature in TMPD-treated mice, which is established within 2 wk of treatment (long before the appearance of anti-nRNP autoantibodies), was abolished in the absence of TLR7. In contrast, the effects of TMPD were amplified in the presence of the Yaa locus. Therefore, this study provides evidence for direct in vivo involvement of TLR7 in the induction of IFN-I, even in the absence of autoantibodies and ICs.
We have previously shown that Ly6Chi
monocytes are a major source of IFN-I in the TMPD model (27
). Depletion of monocytes but not DCs reduced IFN-I production and ISG expression. In this study, we found that Ly6Chi
monocytes also express higher levels of TLR7 and display a greater response in vitro to R848 than to other TLR ligands. Although TLR7 is normally found on monocytes and macrophages, its expression on peritoneal Ly6Chi
monocytes in TMPD-treated mice was several fold higher than on splenic or bone marrow monocytes. In contrast, DCs in the peritoneal exudate displayed lower levels of TLR7 and more prominent TLR3 and TLR9 expression. The high expression level of TLR7 by Ly6Chi
monocytes may be of critical importance in the pathogenesis of TMPD lupus, as a recent study demonstrated that increased gene dosage of TLR7 is sufficient to trigger anti-RNA antibodies and glomerulonephritis in C57BL/6 mice (40
). Interestingly, the recruitment of Ly6Chi
monocytes to the peritoneum seems to be partially dependent on IFN-I, as seen in TLR7−/−
, and IFNAR−/−
mice. TLR7 signaling also induces the expression of several IFN-stimulated chemokines (CCL2
, and CCL12
), suggesting that the mechanism may involve enhanced production of monocyte chemoattractants, creating an amplification loop of Ly6Chi
monocyte recruitment and IFN-I production. The recruitment of DCs and granulocytes, on the other hand, was not dependent on IFN-I or TLR7.
The mechanism linking TMPD to the activation of TLR7 has been partially elucidated by our studies. The hydrocarbon structure of TMPD is distinct from known TLR7 ligands, including ssRNA, R848, loxoribine, and other guanosine analogues. Indeed, our in vitro studies showed that TMPD did not activate TLR7 directly but instead augmented the inflammatory response to TLR7 ligands such as R848. The ability of hydrocarbon oils to enhance TLR7 stimulation in vitro seems related to their ability to induce lupus-like disease in vivo. Unlike TMPD, squalene and medicinal mineral oil were ineffective in augmenting the response to R848. Mice treated with squalene and mineral oil do not display the IFN signature and few develop lupus autoantibodies (27
). TMPD appears to enhance activation via the TLR7 pathway in at least two ways: (a) by augmenting the recruitment of Ly6Chi
monocytes, which express high levels of TLR7, and (b) by enhancing the intrinsic responsiveness of TLR7 to its ligands. Additional studies will be needed to elucidate whether TMPD causes increased uptake of apoptotic/necrotic material, enhancing the recognition of TLR7 ligands, or if TMPD interacts with components of the TLR7 signaling pathway, augmenting the response to receptor–ligand interactions.
Although the underlying mechanisms are distinct, the pathological consequences of excess TLR7 activation are shared by TMPD-induced lupus and the Yaa model. An important unanswered question that encompasses both models is the nature of the exogenous or endogenous ligands responsible for activating the TLR7 pathway. It is possible that chronic TMPD-stimulated inflammation provides a persistent source of autoantigens from apoptotic cells and that endogenous TLR7 ligands such as the U1 RNA component of the Sm/RNP antigen (56
) trigger the first wave of IFN-I production. Downstream signaling events may elicit further IFN-I production (59
) and TLR7
), culminating in a positive feedback cycle that promotes autoimmunity by persistently activating TLR7. It remains to be verified experimentally whether or not RNA-associated autoantigens from apoptotic cells are key mediators of Yaa- and TMPD-induced lupus in vivo.
It is noteworthy that besides activating IFN-I production via TLR7, TMPD also induced the recruitment of granulocytes via an MyD88-dependent but TLR7-independent pathway. The number of peritoneal granulocytes actually increased in the absence of IFN-I production, as seen in TLR7−/− and IFNAR−/− mice. MyD88 is used in the signaling pathways of other cytokines (IL-1 and IL-18) and TLRs (except TLR3), which are potential mediators of granulocyte recruitment in this model.
Finally, our findings may shed light on other pathology induced by TMPD. The development of plasmacytomas after i.p. injection of TMPD in BALB/cAnPt mice was first described more than three decades ago (61
). Subsequently TMPD was used to enhance monoclonal antibody production by hybridomas (62
). How TMPD elicits these effects is incompletely understood, although IL-6 has been implicated. Interestingly, although TLR7 can trigger B cell activation and antibody production (53
), IFN-I plays an important role in antibody class switching and promotes plasma cell differentiation in the presence of IL-6 (63
). Whether TLR7 activation and IFN-I production are involved in the pathogenesis of plasmacytomas and enhancement of antibody production by hybridomas warrants further investigation.