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Abstract
Objective To compare (1) peripheral blood mononuclear cell (PBMC) TLR7 and TLR9 gene expression and (2) interferon stimulated gene (ISG) expression in PBMCs after stimulation with TLR7 and TLR9 agonists between healthy controls (HCs), patients with incomplete systemic lupus erythematosus (iSLE) and patients with SLE.
Methods iSLE patients were included if they had an ANA of ≥ 1:80, and at least one clinical SLICC criterion but less than four criteria in total. All SLE patients met the SLICC criteria for SLE. Gene expression of TLR7 and TLR9 was analysed cross-sectionally with qPCR in PBMCs from 14 HCs, 24 patients with iSLE and 30 patients with SLE. In addition, PBMCs from 10 HC, 30 iSLE and 10 SLE patients were stimulated with TLR7 agonist imiquimod or TLR9 agonist CpG in vitro, and expression of three commonly measured ISGs (MX1, IFI44L, LY6E) was analysed with qPCR. ISG expression was summarized as IFN score using the following formula: ∑(fold induction(ISGsubject)-mean(fold induction(ISGHc))/SD(fold induction(ISGHC))).
Results PBMC TLR7 and TLR9 gene expression was similar between HCs, iSLE and SLE patients (figure 1A-B). Upon stimulation, TLR9 mediated ISG expression was significantly lower in iSLE patients compared to HCs (p<0.001), whereas no significant difference was observed between HCs and SLE patients (figure 1D). In addition, there was a trend for TLR7 mediated ISG expression to be lower in iSLE and SLE patients compared to HCs (p=0.059; figure 1C). TLR9 mediated ISG expression was significantly higher after stimulation in iSLE patients treated with hydroxychloroquine (HCQ) than in patients who were not treated with HCQ (p=0.014; figure 1F). We did not find an association between TLR7 and TLR9 mediated ISG expression and disease activity, autoantibodies or complement levels.
Conclusions TLR7 and TLR9 gene expression in PBMCs was similar between HCs, iSLE and SLE patients. TLR7 and TLR9 mediated ISG response was reduced in PBMCs from iSLE and SLE patients, which might reflect desensitisation due to increased overall TLR7 and TLR9 stimulation. Interestingly, HCQ treatment in iSLE was associated with a higher ISG response in PBMCs upon stimulation, indicating that HCQ might restore normal TLR7 and TLR9 response.
PBMC TLR7 and TLR9 relative gene expression is depicted for HCs, iSLE and SLE patients (A, B). Expression of ISGs, summarized as IFN score, is shown per group after stimulation with TLR7 agonist imiquimod (C) and TLR9 agonist CpG (D). IFN score after stimulation with TLR7 and TLR9 agonists is displayed for iSLE and SLE patients that were or were not treated with hydroxychloroquine (E, F). RE: relative expression; HC: healthy controls; iSLE: incomplete systemic lupus erythematosus, SLE: systemic lupus erythematosus; FI: fold induction; IFN: interferon; ns: non-significant; *: p <0.05; ***: p <0.001, Wilcoxon: Wilcoxon signed rank test.
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