Ongoing IFN-α production in SLE patients appears to play an important pathogenic role in the autoimmune process and pDCs have a pivotal role as the main producers of IFN-α in vivo
]. For this reason, different regimens directed against IFN-α are of high therapeutic benefit in SLE. In this study, we found that pDC production of IFN-α and TNF-α upon TLR-9 or TLR-7 stimulation was markedly reduced in SLE patients treated with HCQ. Although HCQ has been shown to act as a TLR-9 and TLR-7 antagonist in vitro
, these data demonstrate that it also inhibits TLR-9 and TLR-7 stimulation in vivo
. Such inhibition is also specific, in that HCQ treatment has little effect on TLR-4-induced production of TNF-α by monocytes and mDCs. Thus, our data provide new insights regarding the mechanism of action of HCQ in SLE. The reduction of TNF-α TLR-9/7-induced pDC production observed with HCQ is interesting from a therapeutic point of view, even if the involvement of TNF-α in the pathogenesis of SLE remains controversial [28
]. Interestingly, a recent paper has shown that circulating TNF-α and type I IFN levels are correlated in a large cohort of SLE patients [29
]. Finally, similar observations have been made in HCQ-treated HIV-infected "immunologic non-responders" [28
], a condition in which chronic exposure to IFN-α may also lead to immune dysfunction [30
One puzzling and previously reported result is the overall diminished IFN-α production by pDCs in SLE patients compared to controls [11
]. It has been hypothesized that the reduced ability of pDCs to produce IFN-α may be a consequence of the redistribution of the efficient pDCs into tissues [31
] or of the exhaustion of circulating pDCs as a consequence of a high level of stimulation by persistent endogenous IFN-α inducing factors [13
]. Although pDCs are probably migrating to tissues and producing IFN- α there, we did not observe any difference in the percentage of circulating pDCs between SLE patients and healthy controls. We did not either observe any association with IFN-α production by in vitro
stimulated pDCs and SLE disease activity. Kwok et al.
] previously showed that repeated stimulation of pDC with TLR-9 ligand decreased levels of IFN-α production. These authors concluded that the persistent presence of endogenous IFN-α-inducing factors induced TLR tolerance in pDCs of SLE patients. We have now demonstrated that, at least in some patients, such "tolerance" is strongly correlated with the exogenous
administration of HCQ. In fact, most of the SLE patients in previous studies who showed decreased IFN-α production were receiving HCQ [11
]. Our comprehensive study of all subsets of PBMCs potentially targeted by TLR-9/7 ligand also excludes the possibility that other circulating mononuclear cells (besides pDCs) may produce IFN-α. It would have been of interest to determine IFN-α levels and the absolute number of pDCs in the circulation, but such measurements are themselves only uncertain surrogates of the IFN-α and pDC levels in tissues, where most of the pathology of SLE plays out. Although conflicting data may result from the use of different methods to isolate and culture the pDCs between studies, our results support a key role for HCQ treatment in explaining the impaired functional ability of pDCs in SLE.
Low serum HCQ levels have been reported to be a marker for and a predictor of SLE exacerbations [32
], leading to the question of a possible relationship between blood HCQ concentrations and efficacy. We did not observe any significant differences in IFN-α/TNF-α production by TLR-7/9-stimulated pDCs between subjects receiving 400 mg/day and 200 mg/day. We do not know, however, the extent to which each patient was compliant with treatment and serum HCQ levels were not routinely measured.
Lupus activity is commonly measured by valid and reliable disease activity physician-rated scores, such as the Systemic Lupus Activity Measure (SLAM) [33
], the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [34
], or the British Isles Lupus Assessment Group (BILAG) [35
]. All assess cumulative disease activity, including not only parameters that are immunological (for example, the level of antibodies against dsDNA and complement) but also clinical and biological. Practical considerations limited our ability to obtain all of the biological and immunological parameters required to calculate the SLEDAI or BILAG scores.
The SLAQ is a patient-reported assessment of subjective disease activity in SLE that has been shown to correlate strongly (r
= 0.62) with the SLAM-omitting laboratory items [26
] and is considered to be the best measure of self-reported disease activity in the field. It also has the advantage of being the most cost effective way to track disease activity. Using a cutoff score of 13 points on the SLAQ results in a negative predictive value of 74% for clinically important disease activity [26
] defined by a SLAM-omitting laboratory items score ≥3 points. In our study, 30 (77%) subjects had a SLAQ score that was less than 13 and the mean SLAQ score was 10.4. We did not observe an association between TLR-9/7 induced IFN-α production by pDCs and SLE disease activity. However, most of the patients studied here had low levels of disease activity and, because of the number of subjects included, our study may have lacked power to fully address this question.