Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by spontaneous lymphoproliferation, aberrant activation of T and B cells, production of autoantibodies against nuclear DNA and/or nuclear proteins, and formation of immune complex.1,2 Although the etiology of SLE is not fully understood, the mechanism that induces the peculiar autoantibody response in SLE has been explained by recent studies in innate immunity and in particular on the activation of toll like receptors (TLRs) triggered by defective apoptotic cells.3–5
TLRs, known as pattern recognition receptors, play a critical role in protective immunity against invading microorganisms.6 To date, 11 human TLRs have been found, and each of them recognizes unique molecular patterns that are associated with different classes of pathogens, thereby modulating innate and adaptive immunity.7 TLRs have now emerged as important mediators in the pathogenesis of autoimmune diseases.8 Interestingly, animal studies of SLE have implicated that TLR9, a receptor for hypomethylated CpG-containing DNA, plays a crucial role in the pathogenesis of SLE.9,10 Formation of anti-dsDNA autoantibody was mediated by activation of TLR9.9 TLR9 signaling pathway leads to a variety of proinflammatory cytokines and Type I interferon (IFN).11
IFN-α, which belongs to type I IFN, is produced by multiple cell types in response to viral infection. It can exert activities on most major immune cells and function as a bridge between innate and adaptive immunity.12
IFN-α is considered as an important cytokine in the pathogenesis of SLE, because elevated serum levels of IFN-α have been reported in patients with SLE.13 In addition, a significant percentage of patients receiving recombinant IFN-α treatment for cancer or viral hepatitis developed autoantibodies and clinical symptoms that mimic those of SLE.14–16
A common IFN regulatory factor 5 (IRF5) haplotype, driving elevated expression of multiple unique isoforms of IRF5, acts as one of the important transcriptional factors of IFN-α pathway. IRF5 has been confirmed as an important genetic risk factor for SLE.17 In response to TLR9-MyD88 singling pathway, IRF-5 induces the production of inflammatory cytokines, such as IFN-α, IL-6, IL-12, and TNF-α, and subsequently mediates the immune system activation and dysregulation in SLE.18
Recently, three studies in patients with active SLE have also shown an increased expression of TLR9 in peripheral blood mononuclear cells (PBMCs) in Japanese and Greek population.19–21 However, the role of TLR9 is still controversial in lupus.22 Christensen et al9 and Barrat et al23 showed that inhibition of TLR9 resulted in reduction of autoantibody production and amelioration of disease activity. In contrast, studies from two other groups suggested that TLR9 might play a protective role against lupus.24,25 To validate the role of TLR9, we performed the study to investigate the correlation of TLR9 expression with manifestations and laboratory indexes in Chinese SLE patients.
Totally 56 SLE patients were recruited (44 females and 12 males aged (35.4±17.3) years with duration of disease of (6.6±8.6) years) consecutively from the ward of Peking University People's Hospital. All patients fulfilled the American College of Rheumatology (ACR) 1997 revised criteria for SLE. Patients with infection were carefully excluded. Forty healthy age- and gender-matched volunteers (six males and 34 females aged (33.0±12.2) years) were recruited to serve as controls. There is no statistical difference in gender or age between the patient group and the control group (P >0.05). Clinical features and laboratory data were noted from the medical record database of Peking University People's Hospital. Disease activity of SLE patients at the time of visit was evaluated according to Safety of Estrogens in Lupus Erythematosus National Assessment SLE Disease Activity Index (SELENA-SLEDAI). The study protocol was approved by the Ethics Committee of Peking University People's Hospital (FWA00001384) and informed written consent was obtained from all study participants.
Quantitative real time-polymerase chain reaction (qRT-PCR)
PBMCs were isolated from patients with SLE and healthy controls by Ficoll-Histopaque density gradient centrifugation (Sigma, USA). Total RNA was extracted using Trizol® total RNA isolation reagent (Invitrogen, USA) following the manufacturer's protocol. cDNA was made from 1 μg of total RNA in 20 μl reactions and 1.0 μl cDNA was used in the qRT-PCR reaction as described by the manufacturer (Applied Biosystems, USA).
The primer sequences used for the amplification were as follows: GAPDH, forward primer 5′-gacaactttgtatcgtggaaggax-3′ and reverse primer 5′-ggcagggatgatgttctggagag-3′; TLR9, forward primer 5′-cgggtgaagtgtggcagtcc-3′ and reverse primer 5′-gcgagagggcgaaacagtcc-3′. GAPDH was used for normalization (ΔΔCt = ((CtTLR9 - CtGAPDH)sample - (CtTLR9 - CtGAPDH)control). Relative expression levels of TLR9 were determined by using the formula 2-ΔCt. The mRNA level of IRF5 was determined using Taqman® Gene Expression Assay (Part No.: 4331182, ABI company, USA).
Determination of autoantibodies and IFN-α
Serum level of anti-nuclear antibody (ANA) was determined by indirect immunofluorescence (Euroimmun, Germany). Anti-dsDNA antibody was detected by dot immuno-gold filtration assay (Wondofo biomedical Co., China). Levels of anti-histonic antibody (AHA), anti-nucleosome antibody (AnuA), and IFN-α were detected by enzyme-linked immunosorbent assay (ELISA) following the protocol provided by the manufacturer (Euroimmun).
Data analysis was performed using SPSS for Windows software, version 13.0 (SPSS Inc., USA). Correlations between TLR9 expression and disease activity, IRF5 mRNA, and IFN-α expression were analyzed using Spearman's rank correlation test. The statistical significance analysis of TLR9 expression between SLE patients and healthy controls, and between laboratory and clinical indices of SLE patients were performed using Student's t test. P values of less than 0.05 were considered to be significant. Data were expressed as the mean ± standard deviation (SD).
High TLR9 expression in patients with SLE
The mRNA levels of TLR9 expression in PBMCs from 56 SLE patients and 40 control subjects were detected by qRT-PCR. Figure 1 shows that the mRNA level of TLR9 in patients with SLE (2.05±0.18) was significantly higher than that in healthy individuals (1.38±0.16, P=0.0099).
Correlation of TLR9 expression with clinical manifestations of SLE
It is of interest to compare TLR9 mRNA expression levels with clinical manifestations in SLE patients. The results revealed that TLR9 mRNA expression level was significantly positively correlated with fever (P=0.017) and alopecia (P=0.046) in patients with SLE (Table 1). No significant correlation was found between the expression level of TLR9 mRNA and the presence of other clinical manifestations of SLE, such as Raynaud's phenomenon, photosensitivity, rash, oral ulcers, edema, arthralgia, and renal disorder.
TLR9 expression correlated with the disease activity of SLE
To further confirm the role of TLR9 in SLE, we analyzed the correlation of TLR9 mRNA level with SELENA-SLEDAI scores in SLE patients. Expression of TLR9 mRNA level was correlated positively with SELENA-SLEDAI scores (rs=0.385, P=0.003, Figure 2). The serum level of anti-dsDNA antibody in SLE patients tends to reflect disease activity.26 In this study, 29 patients were found to be anti-dsDNA antibody positive, while another 27 patients were negative. Interestingly, TLR9 mRNA expression level in anti-dsDNA antibody positive group was significantly higher compared with that in anti-dsDNA antibody negative group (2.60±1.55 vs. 1.45±0.74, P=0.001, Figure 3). In contrast, there was no significant correlation found between mRNA expression level of TLR9 and the presence of ANA, AnuA, or AHA.
TLR9 expression correlated with IRF5 and IFN-α in SLE
Previous study has shown that TLR9 may induce production of a variety of cytokines, including IFN-α.27 IRF5, an important transcriptional factor, is generally involved in the activation of TLR9 signaling pathway.17 Therefore, we hypothesized that TLR9 expression might be correlated with IRF5 and IFN-α in SLE. We found that TLR9 mRNA expression level was positively associated with mRNA level of IRF5 in the 56 SLE patients (rs=0.35, P=0.027, Figure 4). Furthermore, a positive correlation between TLR9 expression and serum level of IFN-α was observed in patients with SLE (rs=0.627, P=0.001, Figure 5).
Although the etiology of SLE has not been fully understood, there has been intense interest and evidence about the importance of TLRs in the pathogenesis of SLE.3 In this study, we have demonstrated that expression of TLR9 in PBMCs in SLE patients was significantly higher than that in healthy controls. Clinically, expression of TLR9 is significantly associated with disease active index such as SELENA-SLEDAI scores and the presence of anti-dsDNA antibody. Consistent with the finding, increased expression of TLR9 is positively correlated with IRF5 mRNA and IFN-α in patients with SLE. Collectively, these data suggest that activation of TLR9 in SLE might induce upregulation of IRF5 and IFN-α, resulting in deterioration of SLE.
Our results of RT-PCR showed increased expression of TLR9 mRNA level in PBMCs in patients with SLE compared to that in healthy individuals. This is consistent with the previous observations.19–21 SLE patients had higher level of hypomethylated CpG-DNA containing nuclear debris that may serve as autoantigens to trigger TLR9 expression.28,29 Studies in murine lupus models have also suggested that the presence of TLR9 inhibitor can prevent SLE disease development.23,30 Additionally, Epstein-Barr virus infection was identified as one of the potential etiological factors of SLE. TLR9 could be induced and activated by the viral DNA. Thus, the increase of TLR9 expression may be involved in the pathogenesis of SLE.
We found the positive correlation between the TLR9 expression and SLEDAI score. Furthermore, there was a significant positive correlation between TLR9 expression level and fever and alopecia in SLE patients, which are two factors of SLEDAI score. Our data differs from that of Wu et al25 which showed a significantly negative relationship between the level of TLR9 in B cells and SLEDAI score. The discrepancy may be explained by below factors. Firstly we used PBMCs in this study which included more cell types and showed an intact effect of TLR9, while Wu et al used B cells. Secondly, patient selection and composition may differ in these two studies. The discrepancy is not surprising as the role of TLR9 in various murine lupus models is complex and controversial. These apparent contradictions may be explained by different injection schedules and adjuvants. In contrast, in the same murine model, the use of inhibitory immunoregulatory DNA sequences to inhibit TLR7 and TLR9 improved the disease, decreasing the autoantibody titers, improving renal function, and prolonging survival. In humans, TLR7 and TLR9 inhibition by IRS 954 prevented the IFN-α production.23 These findings are consistent with our data that upregulated expression levels of TLR9 mRNA was associated with enhanced anti-dsDNA antibody and SELENA-SLEDAI scores.
Anti-dsDNA autoantibody production is a hallmark of disease activity for SLE. In our patients, we found positive correlation between increased expression of TLR9 mRNA and the presence of anti-dsDNA antibody. This is consistent with the studies by Christensen et al9 which showed that the production of anti-dsDNA antibodies was inhibited in TLR9-deficient lupus-prone mice. This suggests that TLR9 is required for the generation of anti-dsDNA antibody. One possible mechanism is that autoantigens can bind to BCRs and trigger TLR9 expression, which results in B cell activation, proliferation, and autoantibody production.31 In addition, activation of TLR9 leads to the production and class-switching of antibodies.32,33 Therefore, the TLR9 antagonist can block the TLR9 signaling to prevent anti-dsDNA antibody production and ameliorate the disease. The TLR9 signaling pathway could be activated, resulting in anti-dsDNA antibody production which is involved in the pathogenesis and disease. In contrast, in anti-dsDNA antibody negative cases, some alternative mechanisms such as other TLRs might be involved in the development of SLE.34
Previous studies have demonstrated that activation of TLR9 could provoke antigen presentation of dendritic cells (DCs) and enhance cytokine production, including IFN-α, thereby causing tissue injury and inflammation.9,10,19 We observed that TLR9 mRNA expression level was positively correlated with serum IFN-α level. IFN-α is mainly produced by activated DCs. Immune complex containing nucleic acids is internalized by plasmacytoid DCs (pDCs) via cell surface Fcγ RIIa and relocated to endosomal compartment where they engage TLR9.13 This consequently leads to the activation and secretion of proinflammatory cytokines including IFN-α by DCs.12,13 It is therefore suggested that TLR9 plays a critical role in the development of SLE through the induction of IFN-α. In addition to virus clearance, IFN-α also plays an important role in promoting polyclonal T cell activation and response, induces class-switching of immunoglobulin, enhances expression of MHC II molecules, and results in differentiation of DCs, thereby contributing to the pathogenesis of SLE.35 Studies have shown that IFN-α correlates positively with disease activity in SLE, which is further supported in our study.
IRF5 is clearly documented in the downstream of TLR9-MyD88 signaling pathway in the induction of proinflammatory cytokines, such as IFN-α, IL-6, IL-12, and TNF-α.35 Recent advances in genetic analysis techniques have identified IRF5 as one of the human lupus-associated genes. In pristane-induced murine SLE model, IRF5 is shown to be essential for the development of ANAs and immune complex glomerular deposits.36 The implication of IRF5 in SLE patients may further indicate the role of TLR9 in the pathogenesis of human lupus. Our study showed for the first time, that increased expression of TLR9 mRNA level was associated with increased expression of IRF5 mRNA level. This result indicates the important role of TLR9 for its potential regulation of IRF5 expression in SLE patients.
In conclusion, there is a positive correlation between the TLR9 levels and clinical and laboratory indexes in SLE patients. Our study suggests that expression of TLR9 might be involved in the pathogenesis of SLE and a potential biomarker for the disease activity.
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