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Author(s) : N. R. Ghaly et al.

Article title : Toll-like receptor 9 in systemic lupus erythematosus, impact on glucocorticoidtreatment

Article no : 697110

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1 Nahla R. Ghaly

2 Nesreen A. Kotb

3 Hala M Nagy

4 El Sayed M Rageh

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Journal of Dermatological Treatment, 2012; Early Online: 1–7© 2012 Informa Healthcare USA on behalf of Informa UK Ltd.ISSN: 0954-6634 print / 1471-1753 onlineDOI: 10.3109/09546634.2012.697110

1 ORIGINAL ARTICLE

2 Toll-like receptor 9 in systemic lupus erythematosus, impact on3 glucocorticoid treatment

4 Nahla R. Ghaly1, Nesreen A. Kotb2, Hala M Nagy3 & El Sayed M Rageh4

5 1Tanta University, Faculty of Medicine, Dermatology & Venereology, Tanta, Egypt, 2Tanta University, Faculty of Medicine, Internal Medicine,6 Tanta, Egypt, 3Tanta Unversity, Faculty of Medicine, Clinical Pathology, Tanta, Egypt and 4Tanta University, Faculty of Medicine, Physical7 Medicine, Rheumatolgy and Rehabilitaion, Tanta,AQ3 Egypt

8910 Aim: To assess TLR9 expression in systemic lupus erythematosus11 (SLE) patients, its correlation with disease activity, and impact of12 TLR9 expression on the response to oral glucocorticoids. Methods:13 Twenty-five active SLE, 15 inactive, and 15 control subjects were14 included. Anti-DNA, ANA, C3, C4, and TLR9 mRNA expressions15 were assessed. Active SLE patients only received oral steroid for16 6 weeks. Post therapy, they were classified into steroid sensitive17 and steroid resistant. Data were reassessed after treatment.18 Results: SLEDAI, anti-DNA, ANA, and TLR9 expressions were19 significantly higher in active SLE patients. Based on retrograde20 analysis, TLR9 expression was significantly higher in steroid-21 resistant versus steroid-sensitive group before treatment, with22 no significant difference between them after treatment. There was23 a significant positive correlation between TLR9 expression and24 SLEDAI score and anti-DNA and negative correlation with C3 and25 C4 in all patients. Conclusion: TLR9 may play a role in the26 pathogenesis of SLE and correlates with the disease activity.27 Corticosteroids have no effect on TLR9 expression, explaining28 lack of corticosteroid response in some SLE patients. TLR 929 expression can be used in predicting glucocorticoid response in30 active SLE patients. New treatment modalities targeting31 TLR9 expression may be of value in steroid-resistant patients.3233 Key words: autoimmunity, expression, B cell, steroid resistant

34 Introduction

35 Systemic lupus erythematosus (SLE) is a chronic inflammatory36 disease of generalized autoimmunity, which is characterized by37 B-cell hyperactivity, abnormally activated T cells, and defects in38 the clearance of apoptotic cells and immune complexes (1).39 Several mechanisms have been proposed to explain the pro-40 duction of autoantibody-producing B cells: impaired survival/41 apoptosis signals preventing negative selection, dysfunctional42 complement, or inhibitory Fc receptors, loss of peripheral43 tolerance through activation of myeloid dendritic cells induced44 by interferon (IFN)-a overproduction and activation of Toll-45 like receptors (TLRs) in response to accumulation of apoptotic46 bodies (2).

47TLRs constitute an important part of the group of glycoprotein48receptors that recognize these molecular patterns called “pattern49recognition receptors” (PRRs) questioning the nonspecific nature50of the innate immune response system (3). Moreover, they can51participate in B-cell differentiation and immunoglobulin produc-52tion in a T-cell-independent manner. In the last several years, it53has become apparent that TLRs can participate in cell activation54by self-molecules such as immune complexes containing DNA or55RNA (4).56Toll-like receptor 9 (TLR9) has recently been implicated in the57activation of autoreactive B cells in murine models of SLE. Its58recognition of CpG nucleotide sequences appears to enhance the59activation of B cells via the B-cell receptor. Based on this, it has60been demonstrated that the dual engagement of the B-cell recep-61tor (BCR) and TLR9 by DNA-containing immune complexes may62prime autoantibody production by native B cells (5). Subsequent63studies in humans have shown that dendritic cells are activated by64chromatin immune complexes and that this effect is mediated by65cooperation between TLR9 and Fc_ receptors. These observations66suggest that genetic variation affecting the costimulatory function67of TLR9 could lead to differences in B-cell response to autoanti-68gens and dendritic cell response to chromatin immune complexes.69Thus, a great deal of work has been directed toward understand-70ing how these receptors act in disease progression and the71relevance of these findings to the clinical manifestations and72activity of SLE (6).73Glucocorticoid (GC) therapy is the main treatment for SLE.74However, some patients are resistant to these agents, so-called75steroid-resistant patients who fail to respond to treatment with76glucocorticoids (7). Moreover, glucocorticoids do not significantly77reduce the production of IFN-a upon plasmacytoid dendritic cell78(PDC) activation with the TLR9 ligands influenza virus (FLU)79or with immune complexes from SLE patients (8). More aggres-80sive approaches such as methylprednisolone pulse therapy or81immunosuppressive drugs are needed in such patients to control82disease activity, the pathogenic mechanism of which is not fully83understood (9).84In the present study, we have examined the level of TLR859 expression on whole blood samples in patients with active86SLE versus inactive SLE patients and its correlation with disease

Correspondence: Nahla R. Ghaly, MSc MD, Tanta University, Faculty of Medicine, Dermatology & Venereology, Tanta, Egypt. Tel: +0096597387170.E-mail: nrghaly@yahoo.com

(Received 11 January 2012; accepted 30 April 2012)

87 activity parameter. We also assessed the impact of TLR9 expres-88 sion in active patients over their response to oral glucocorticoid89 treatment.

90 Material and method91 We recruited 25 newly diagnosed untreated active SLE patients,92 15 sex and age matched with inactive SLE under maintenance93 therapy with only corticosteroid who were at regular follow-up visit94 and 15 sex- and age-matched healthy subjects as a disease control95 from outpatient clinic of Internal medicine and dermatology96 departments, Tanta university hospital, Egypt. All patients fulfilled97 the American College of Rheumatology (ACR) 1997 revised criteria98 of SLE (10). Disease activity index was assessed by using the ACR99 SLE disease activity index (SLEDAI) (11).100 For all participants, history taking, clinical examination, ANA,101 anti-double-stranded DNA, and anti-Sm antibodies C3 and102 C4 were assessed. We quantified Toll-like receptor 9 mRNA103 expression for SLE patients (active and nonactive) and healthy104 subjects.105 Patients with active SLE (25 patients) received only oral steroid106 therapy (dose 1–2 mg/kg) for 6 weeks. Post therapy, they were107 classified into steroid sensitive and steroid resistant according to108 their post-therapy SLEDAI score. Decrease of SLEDAI score109 ‡4 from their pretreatment score were considered as steroid110 sensitive. For both steroid sensitive and steroid resistant, laboratory111 data and Toll-like receptor 9 mRNA expression were re-assessed.112 The protocol of the study was approved by the research113 committee of the Faculty of Medicine, Tanta university hospital.114 Informed written consent prior to participation in the study was115 obtained from all patients and healthy subjects.

116 Sample collection

117 Blood samples.118 Blood samples (7 ml) were obtained from patients and controls;119 3 ml of the sample was collected to an EDTA tube for performing120 DNA extraction and 2 ml in another tube to perform complete121 blood picture. The rest of the samples were centrifuged at

1223000 � g for 10 min at 4�C, after that serum was removed123and used for determination of C3 and C4 by nephelometry124(Behring GmbH, Marburg, Germany) (12), ANA, anti-DNA,125and anti-Sm antibodies were determined by enzyme-linked126immunosorbent assays (Orgentec Diagnostika GmbH, Mainz,127Germany). Urine analysis, 24-h urinary protein excretion128(UPE) (13), and creatinine clearance (14) were determined.

129RNA extraction130Extraction of RNA was done from blood samples by Qiagen131RNeasy Mini Kit according to the protocol supplied by the132manufacturer. One volume of 70% ethanol was then added to133the cleared lysate and mixed immediately, then transferred to134Qiagen RNeasy spin column placed in a 2-ml collection tube and135proceeded according to the manufacturer’s instructions. RNA was136eluted and its concentration was measured spectrophotometri-137cally (280). Five hundred nanogram of total RNA was used for138first-strand cDNA synthesis primed with 50 pmol of random139primer (Roche, Mannheim, Germany) and 0.5 ml (200 U/ml)140reverse transcriptase Superscript� II-RT (Invitrogen, Karlsruhe,141Germany). PCR cycling condition was performed according to142the manufacturer’s instructions.

143Reverse transcriptase – PCR (15).144One microliter of the reverse transcriptase reaction mixture was145added to a 20-ml reaction mixture of the QuantiTect SYBR-Green

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Figure 1. Amplification plot of TLR9 and GAPDH showing the log of the change in the fluorescence plotted against cycle number.

Table I. Clinical characteristics of systemic lupus erythematosus (SLE)patients.

SLE group (40)

Variables Number %

Arthritis 24 60Serositis 10 25Vasculitis 11 27.5Renal involvement 7 17.5Hematological manifestation 17 42.5Fever 23 57.5Mucosal ulcer 15 37.5

N. R. Ghaly et al.

146 PCR kit (Qiagen) and 0.5 mM from the specific primer pair for147 human TLR9-forward, 5-TAG AGA CTT CAG GCC CAA148 CTG-3, and TLR9-reverse, 5-TGC ACG GTC ACC AGG TTG149 T-3. Primers for GAPDH are GAPDH-forward ATG GCT ATG150 ATG GAG GTC CAG and GAPDH-reverse, TTG TCC TGC151 ATC TGC TTC AGC. A negative control containing the complete152 Master Mix without a DNA template was included. Samples of153 cDNA were assayed on the step one Real Time PCR System

154(Applied Biosystems). Initial denaturation at 95�C for 900 sec was155followed by 45 cycles with denaturation at 95�C for 15 sec, anneal-156ing at 64�C for 20 sec and extension at 72�C for 20 sec. The157fluorescence intensity of SYBR-Green, specifically incorporated in158the double-stranded DNA amplicon reflecting the amount of159formed PCR product was read after each extension step at16072�C. RNA amounts were determined with the Applied Biosys-161tems software in a mode relative to GAPDH gene (Figure 1).

Table II. Comparison between all studied groups with regard to the clinical and biochemical parameters

SLE (40) ANOVA Tukey’s test

Variable Control 15 Inactive 15 Active (25) F p-value I&II I&III II&III

AQ8 SLEDAI ScoreMean 3.50 21.15 13.76t < 0.001SD 1.83 4.58

Corticosteroid doseMean 11.25 60.55 33.472t < 0.001SD 2.55 5.33

ANA (IU/ml)Mean 6.43 21.89 25.66 268.1 < 0.001 <0.001 <0.001 <0.001SD 3.35 2.61

Anti-DNA (IU/ml)Mean 1.58 81.70 171.6 < 0.001 <0.001 <0.001 <0.001SD 5.70 36.10 9.02

Anti-SM (IU/ml)Mean 10.55 12.55 29.84 7.8 0.001 0.949 <0.001 <0.001SD 1.53 1.21 3.66

C3 (mg/dl)Mean 146.30 138.81 58.23 208.9 < 0.001 0.393 <0.001 <0.001SD 27.29 7.32 6.62

C4 (mg/dl)Mean 35.22 32.56 9.10 595.5 < 0.001 0.26 <0.001 <0.001SD 3.97 2.23 1.85

TLR9 expressionMean 1.61 1.88 6.41 250.5 < 0.001 0.597 <0.001 <.001SD 0.74 1.00 0.65

Table III. The clinical and biochemical parameters before and after treatment within the same group.

Before After

Mean SD Mean SD % of change Paired t-test

SLEDAI ScoreSteroid sensitive 18.4 3.7 10.6 2.65 –42.391 0.010Steroid resistant 25.5 1.3 29.3 1.88 14.902 0.031

ANA (IU/ml)Steroid sensitive 25.3 2.8 19.2 3.02 –24.111 0.022Steroid resistant 26.3 2.3 28.6 2.12 8.745 0.039

Anti-DNA (IU/ml)Steroid sensitive 76.7 6.7 57.3 5.55 –25.293 0.000Steroid resistant 89.8 5.9 105.6 4.66 17.595 0.000

Anti-SM (IU/ml)Steroid sensitive 23.6 3.54 22.7 2.55 –3.814 0.512Steroid resistant 25.66 3.55 24.52 4.12 –4.443 0.772

C3 (mg/dl)Steroid sensitive 61.8 5.3 93.2 4.55 50.809 0.000Steroid resistant 52.5 3.9 49.72 2.78 –5.295 0.128

C4 (mg/dl)Steroid sensitive 10.3 1.1 21 2.05 103.883 0.000Steroid resistant 7.2 1.1 6.93 1.82 –3.750 0.243

TLR9 expressionSteroid sensitive 6.03 0.49 5.9 0.56 –2.156 0.096Steroid resistant 7.01 0.33 7.37 0.46 5.136 0.722

TLR9 in SLE, impact on glucocorticoidAQ2 treatment

162 Statistical analysis163 Data were analyzed with the GraphPad InStat. Descriptive data were164 given as mean ± standard deviation (SD). Comparison between the165 two groups was performed with t-test, but among different groups,166 ANOVA was preformed followed by Tukey–Kramer Multiple com-167 parisons test. A spearman rank correlation was performed to deter-168 mine the associations between the studied parameters and TLR9. A p169 value less than 0.05 was considered statistically significant.

170 Results171 This study was performed on 25 patients with newly diagnosed172 active SLE (F/M was 20:5) with SLEDAI score >4 and 15 patients173 with inactive active SLE (F/M was12:3) with SLEDAI score £4.174 Fifteen sex- and age-matched healthy subjects (F/M was12:3)175 were also included as control group. Table I shows clinical176 characteristics of SLE patients.177 The SLEDAI scorewas significantly higher in active group versus178 inactive group (p < 0.001). Corticosteroidmaintenance dose ranged179 from 5 to 20 mg/day in inactive group while it was 50–80 mg/180 day in active group with (p < 0.001). ANA and anti-DNA were181 significantly higher in inactive and active groups as compared with182 the control group (p < 0.001). The serum concentration of C3 and

183C4were significantlydecreased inactivegroupas comparedwith the184inactive and control groups (p<0.001)while therewasnosignificant185difference between inactive and control groups (p > 0.05). Anti-186SM and TLR9 expression were significantly higher in the active187group as compared with the inactive and control groups (p < 0.001)188while there was no significant difference between inactive and189control groups (p > 0.05) (Table II).190Based on retrograde analysis, there was no significant dif-191ference between steroid-sensitive and steroid-resistant group192regarding corticosteroid dose received (p > 0.05). Table III193shows comparison of clinical and biochemical parameters194before and after treatment within steroid-sensitive and195steroid-resistant groups separately. SLEDAI score, ANA, and196anti-DNA were significantly decreased in steroid-sensitive197group after treatment while they were significantly increased198steroid-resistant group after treatment. The serum concentra-199tion of C3 and C4 was significantly increased in steroid-200sensitive group after treatment (p < 0.001) while there was201no significant difference of their values after treatment in202steroid-resistant group (p > 0.05). There was no significant203difference of TLR9 expression value within the same group204after treatment versus before treatment (p > 0.05).205Table IV shows that SLEDAI score, and anti-DNA were206significantly higher in steroid-resistant group when compared207with steroid-sensitive group either before or after treatment208(p < 0.001). Regarding ANA, there was no significant difference209between steroid-sensitive and steroid-resistant group before treat-210ment (p > 0.05), while after treatment, ANAwas significantly higher211in steroid-resistant group when compared with steroid-sensitive212group (p < 0.001). Anti-SM shows no significant difference between213steroid-sensitive and steroid-resistant groups either before or after214treatment (p > 0.05). The serum concentration of C3 and C4 were215significantly higher in steroid-sensitive group when compared with216steroid-resistant group either before treatment or after treatment217(p < 0.001).

Table IV. Comparison between steroid-sensitive and steroid-resistant groups with regard to the clinical and biochemical parameters before and aftertreatment.

Steroid sensitive Steroid resistant T-test

Mean SD Mean SD t p-value

SLEDAI ScoreBefore 18.4 3.7 25.5 1.3 6.882 0.000After 10.60 2.65 29.30 1.88 20.694 0.000

ANA (IU/mL)Before 25.3 2.8 26.3 2.3 0.977 0.338After19.20 3.02 28.6 2.12 7.609 0.000

Anti-DNA (IU/mL)Before 76.7 6.7 89.8 5.9 5.155 0.000After 57.30 5.55 105.60 4.66 23.535 0.000

Anti-SM (IU/mL)Before 23.6 3.54 25.66 3.55 1.423 0.168After 22.70 2.55 24.52 4.12 1.242 0.227

C3 (mg/dl)Before 61.8 5.3 52.5 3.9 5.062 0.000After 93.20 4.55 49.72 2.78 26.943 0.000

C4 (mg/dl)Before 10.3 1.1 7.2 1.1 6.903 0.000After 21.00 2.05 6.93 1.82 17.555 0.000

TLR9 expressionBefore 6.03 0.49 7.01 0.33 5.997 0.000After 5.90 0.56 7.37 0.46 7.180 0.000

Table V. Correlation between the TLR9 expression and other variables in allsystemic lupus erythematosus (SLE) patients.

TLR9 expression

Variables r p-value

Score 0.625 0.021Corticosteroid dose 0.125 0.890ANA (IU/ml) 0.241 0.688Anti-DNA (IU/ml) 0.651 0.021Anti-SM (IU/ml) 0.116 0.900C3 (mg/dl) –0.771 0.001C4 (mg/dl) –0.702 0.005

N. R. Ghaly et al.

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221 TLR9 expression was significantly higher in steroid-resistant222 versus steroid-sensitive group either before treatment or after223 treatment (p < 0.001). There was a significant positive correla-224 tion between TLR9 expression and SLEDAI score and Anti-225 DNA (IU/mL) (p < 0.05). Also, there was a significant negative226 correlation between TLR9 expression and serum concentration of227 C3 and C4 (p < 0.05) while there was no correlation between228 TLR9 expression and anti-SM (p > 0.05) (Table V).229 Using the ROC curve, TLR9 expression level >6.54 was esti-230 mated to be the cut-off value for detection of non-responder in231 active SLE patients with 0.738% accuracy, 85.7 negative predictive232 value, 66.7% positive predictive value, 80.0 sensitivity, and 75.0%233 specificity (Table VI).

234 Discussion235 Over the last several years, new emphasis has been placed on236 the role of the innate immune system in amplification of auto-237 immune response and exacerbation of SLE. Toll-like receptors238 have been recognized as essential component in innate immunity239 and promote the generation of autoantibodies against nuclear240 components.241 In the present study, TLR9 was significantly higher in patients242 with active SLE compared with patients with inactive SLE and243 controls and it correlated positively with ANA, anti-DNA, and244 SLEDAI score. Our results support the previously published245 studies that raise the possibility of TLR9 having a role in the246 pathogenesis of SLE. A study by Nakano et al. (16) demonstrated247 that higher expression of TLR9 on peripheral blood B cells from248 patients with active SLE was significantly correlated with SLEDAI249 and induced anti-dsDNA antibody and IL-10 through ligation of250 CpG to TLR9 on B cells. Using cytoplasmic staining and RT-PCR,251 they confirmed that the expression of TLR9 was increased on B252 cells from patients with active SLE, and this was correlated with253 disease activity. Furthermore, deficiencies of TLR9 suppress the254 production of anti-RNA or anti-DNA autoantibodies in murine255 models of SLE (17).256 TLR induced the expression of co-stimulatory molecules, as257 represented by CD80 and CD86, and the expression of TLR9 was

258correlated with that of CD86 on B cells. Since the expression of259CD86 on B cells is commonly increased in active SLE, it is possible260that the increased expression of TLR9 induces the initial activa-261tion of B cells. Thus, TLR9 expression on B cells plays a role in262SLE pathogenesis through the induction of anti-dsDNA antibody.263Moreover, methylation of DNA is decreased in SLE patients,264suggesting that the interaction between CpG and TLR9 plays a265pivotal role in anti-dsDNA antibody production (18).266Several studies have demonstrated that anti-nucleic acid267immune complexes, isolated from the sera of SLE patients, stim-268ulate plasmacytoid dendritic cells (pDCs) to produce IFN-a269(19,20). Mean et al. (21) have reported that DNA-containing270immune complexes, but not protein-containing immune com-271plexes, stimulate pDCs to produce cytokines, including IFN-a,272TNF, and IL-8. This process is thought to be mediated by a273cooperative interaction between TLR9 and FcgRIIa, supporting274the “dual receptor paradigm” in promoting activation of the275innate immune system. Barrat et al. (22) showed that mammalian276RNA and DNA, in the form of immune complexes, are potent277self-antigens for TLR7 and TLR9, respectively, and induce278IFN-a production by plasmacytoid dendritic cells.279In the present study, there was a significant positive correlation280between TLR9 expression and SLEDAI score and anti-DNA. Also281there was a significant negative correlation between TLR9 expres-282sion and serum concentration of C3 and C4. Recently, two study283groups examined the expression of TLRs in peripheral blood284mononuclear cells (PBMCs) from SLE patients by flow cytometry285(6,23). It has been demonstrated that the proportion of plasma cells286and memory B cells expressing TLR9 was increased in active SLE287patients and correlated with the presence of anti-dsDNA anti-288bodies in their patients, which is in accordance with our finding289(6). In fact, patients with active SLE have increased expression of290TLR9 in peripheral blood memory and plasma B lymphocytes,291suggesting that endogenous nucleic acids released during apoptosis292may stimulate B lymphocytes via TLR9 and contribute to SLE293pathogenesis (24). In contrast to our finding, Migita et al. (23) also294showed no significant correlation between TLR9 expression levels295and clinical markers such as SLEDAI scores and anti-dsDNA296antibodies in their patients. In experimental studies of murine297lupus, there were also opposite findings regarding the role of298TLR9 in anti-DNA antibody formation (25,26). This discrepancy299might be due to the different genetic background of mice used in300these studies.301To the best of our knowledge, our study is the first one302evaluating the relation between TLR9 expression and response303to oral glucocorticoids therapy in active human SLE patients.304In our study, 15 patients with active SLE were steroid sensitive305with significant post-therapy decrease in their SLEDAI score,306while the other 10 patients were steroid resistant with significant307post-therapy increase in their SLEDAI score. By retrograde308analysis of our patients clinical and laboratory data before therapy,309SLEDAI score and anti-DNA were significantly higher, C3 and C4310were significantly lower in steroid-resistant group when compared311with steroid sensitive. TLR9 expression was significantly higher in312steroid resistant versus steroid sensitive either before treatment or313after treatment. TLR9 expression did not change significantly314either in steroid-sensitive or steroid-resistant group upon treat-315ment. Thus, these data suggest that assessment of TLR9 expression316can be used in predicting glucocorticoid treatment response in317active SLE patients.318Glucocorticoids induce apoptosis in many cell types, including319PDCs, where TLR signaling confers partial protection (7). Freshly320isolated PDCs from healthy donors stimulated with TLR9 ligands321were protected from glucocorticoid-induced cell death. This dose-

Table VI. ROC curve for detection of cut-off value of TLR9 expression indetecting non-responder among active systemic lupus erythematosus (SLE)patients.

ROC curve

Cut-off Sens. Spec. PPV NPV Accuracy

>6.54 80.0 75.0 66.7 85.7 0.738

TLR9 in SLE, impact on glucocorticoidAQ2 treatment

322 dependent protection correlated with the production of IFN-a by323 PDCs supporting our data at a single cell level (27). Blocking this324 pathway with IRS 954 (bifunctional TLR9 inhibitor called immu-325 noregulatory sequence) restored glucocorticoid sensitivity to326 PDCs in vitro although IRS 954 itself was not cytotoxic (22).327 Moreover, signaling through TLR9 protects human PDCs from328 glucocorticoid-induced cell death. Taken together, the human329 data may be interpreted as being consistent with the experimental330 mouse studies and support the concept that TLR9 activation331 by DNA-containing immune complexes is crucial to SLE332 pathogenesis (28).333 Guiducci et al. (29) reported that in vitro and in vivo334 stimulation of PDCs through TLR9 can account for the reduced335 activity of glucocorticoids to inhibit the IFN pathway in SLE336 patients and in two lupus-prone mouse strains. The triggering of337 PDCs through TLR9 by nucleic acid-containing immune com-338 plexes or by synthetic ligands activates the NF-kB pathway339 essential for PDC survival. They added that glucocorticoids do340 not affect NF-kB activation in PDCs, preventing glucocorticoid341 induction of PDC death and the consequent reduction of342 systemic IFN-a level. These findings unveil a new role for343 self nucleic acid recognition by TLRs and indicate that inhibi-344 tors of TLR9 signaling could prove to be effective corticosteroid-345 sparing drugs. In experimental study (30), TLR9 activation with346 CpG-ISS (CPG immunostimulatory sequence) afforded signif-347 icant protection from glucocorticoid-induced cell death to

AQ4348 conventional and PDCs in both spleen and blood. Circulating349 PDCs are significantly more susceptible to glucocorticoid-350 induced cell death than TLR-activated PDCs in vivo. In addi-351 tion, treatment of lupus-prone mice with a dual inhibitor of352 TLR9 leads to the reduction of autoantibody production and353 amelioration of disease symptoms. Although the direct TLR354 antagonists have not been studied in human patients, inhibitors355 of IFN-a, a primary downstream effector of TLR signaling and356 important disease mediator in SLE, have been developed and are357 currently undergoing clinical trials (31).358 As a consequence, the accumulation of evidence pointing359 toward TLRs in autoimmunity has opened the door for potential360 therapeutic interventions directed toward the modulation of361 Toll-like receptors and their signaling pathway. Since there362 are several proteins involved in TLR signaling, there are a363 number of targets that may be utilized for potential drugs.364 Some of the possibilities include development of TLR antago-365 nists, inhibitors of downstream signaling events, activation of366 natural inhibitory molecules, or blockade of the effector mole-367 cules produced (32). Although there has not been a new drug368 approved for the treatment of lupus in many years, current369 investigation regarding the targeting of TLRs and their down-370 stream effectors is showing some promise and warrants high371 expectations.

372 Conclusions373 TLR9 may play a role in the pathogenesis of SLE and correlates374 with the disease activity. Corticosteroids have no effect on TLR9375 expression and this can explain lack of corticosteroid response in376 some of SLE patients. TLR9 expression can be used in predicting377 glucocorticoid treatment response in active SLE patients. New378 modalitiesof treatment targetingTLR9expressionandantagonizing379 their effect will be of value in SLE steroid-resistant patients.

380 Declaration of interest: The authors report no conflicts of381 interest. The authors alone are responsible for the content and382 writingAQ5 of the paper.

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460 26. Wu X, Peng SL. Toll-like receptor 9 signaling protects against murine461 lupus. Arthritis Rheumatol. 2006;54:336–342.462 27. Boor PP, et al. Prednisolone suppresses the function and promotes463 apoptosis of plasmacytoid dendritic cells. Am J Transplant. 2006;6:464 2332–2341.465 28. Guiducci C. PI3K is critical for the nuclear translocation of IRF-7-466 and type I IFN production by human plasmacytoid467 predendritic cells in response to TLR activation. J Exp Med. 2008;468 205:315–322.469 29. Guiducci C, GongM, Xu ZA, et al. TLR recognitionAQ6 of self nucleicAQ7 acids470 hampers glucocorticoid activity in lupus. Nature. 2011;1–10.

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TLR9 in SLE, impact on glucocorticoidAQ2 treatment

1

Original Article

Toll-like receptor 9 in systemic lupus erythematosus, impact on glucocorticoid treatment

TLR9 in SLE, impact on glucocorticoid treatment

N. R. Ghaly et al.

Nahla R. Ghaly1, Nesreen A. Kotb2, Hala M Nagy3 & El sayed M Rageh4 1Tanta University, Faculty of Medicine, Dermatology & Venereology, Tanta, Egypt 2Tanta University, Faculty of Medicine, Internal Medicine, Tanta, Egypt Tanta Unversity, Faculty of Medicine, Clinical Pathology, Tanta, Egypt 4Tanta University, Faculty of Medicine, Physical Medicine, Rheumatolgy and Rehabilitaion, Tanta, Egypt Correspondence: Nahla R. Ghaly, MSc MD, Tanta University, Faculty of Medicine, Dermatology & Venereology, Tanta, Egypt. +0096597387170. nrghaly@yahoo.com

Aim:

To assess TLR9 expression in systemic lupus erythematosus (SLE) patients, its correlation with disease activity, and impact of TLR9 expression on the response to oral glucocorticoids.

Methods:

Twenty-five active SLE, 15 inactive, and 15 control subjects were included. Anti-DNA, ANA, C3, C4, and TLR9 mRNA expressions were assessed. Active SLE patients only received oral steroid for 6 weeks. Post therapy, they were classified into steroid sensitive and steroid resistant. Data were reassessed after treatment.

Results:

SLEDAI, anti-DNA, ANA, and TLR9 expressions were significantly higher in active SLE patients. Based on retrograde analysis, TLR9 expression was significantly higher in steroid-resistant versus steroid-sensitive group before treatment, with no significant difference between them after treatment. There was a significant positive correlation between TLR9 expression and SLEDAI score and anti-DNA and negative correlation with C3 and C4 in all patients.

Conclusion:

TLR9 may play a role in the pathogenesis of SLE and correlates with the disease activity. Corticosteroids have no effect on TLR9 expression, explaining lack of corticosteroid response in some SLE patients. TLR 9 expression can be used in predicting glucocorticoid response in active SLE patients. New treatment modalities targeting TLR9 expression may be of value in steroid-resistant patients.

Key words: autoimmunity, expression, B cell, steroid resistant

2

Introduction

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease of generalized

autoimmunity, which is characterized by B-cell hyperactivity, abnormally activated T cells,

and defects in the clearance of apoptotic cells and immune complexes 1. Several mechanisms

have been proposed to explain the production of autoantibody-producing B cells: impaired

survival/apoptosis signals preventing negative selection, dysfunctional complement, or

inhibitory Fc receptors, loss of peripheral tolerance through activation of myeloid dendritic

cells induced by interferon (IFN)-α overproduction and activation of Toll-like receptors

(TLRs) in response to accumulation of apoptotic bodies 2.

TLRs constitute an important part of the group of glycoprotein receptors that recognize these

molecular patterns called “pattern recognition receptors” (PRRs) questioning the nonspecific

nature of the innate immune response system 3. Moreover, they can participate in B-cell

differentiation and immunoglobulin production in a T-cell-independent manner. In the last

several years, it has become apparent that TLRs can participate in cell activation by self-

molecules such as immune complexes containing DNA or RNA 4.

Toll-like receptor 9 (TLR9) has recently been implicated in the activation of autoreactive B

cells in murine models of SLE. Its recognition of CpG nucleotide sequences appears to

enhance the activation of B cells via the B-cell receptor. Based on this, it has been

demonstrated that the dual engagement of the B-cell receptor (BCR) and TLR9 by DNA-

containing immune complexes may prime autoantibody production by native B cells 5.

Subsequent studies in humans have shown that dendritic cells are activated by chromatin

immune complexes and that this effect is mediated by cooperation between TLR9 and Fc_

receptors. These observations suggest that genetic variation affecting the costimulatory

function of TLR9 could lead to differences in B-cell response to autoantigens and dendritic

3

cell response to chromatin immune complexes. Thus, a great deal of work has been directed

toward understanding how these receptors act in disease progression and the relevance of

these findings to the clinical manifestations and activity of SLE 6.

Glucocorticoid (GC) therapy is the main treatment for SLE. However, some patients are

resistant to these agents, so-called steroid-resistant patients who fail to respond to treatment

with glucocorticoids 7. Moreover, glucocorticoids do not significantly reduce the production

of IFN-α upon plasmacytoid dendritic cell (PDC) activation with the TLR9 ligands influenza

virus (FLU) or with immune complexes from SLE patients 8. More aggressive approaches

such as methylprednisolone pulse therapy or immunosuppressive drugs are needed in such

patients to control disease activity, the pathogenic mechanism of which is not fully

understood 9.

In the present study, we have examined the level of TLR 9 expression on whole blood

samples in patients with active SLE versus inactive SLE patients and its correlation with

disease activity parameter. We also assessed the impact of TLR9 expression in active patients

over their response to oral glucocorticoid treatment.

Material and method

We recruited 25 newly diagnosed untreated active SLE patients, 15 sex and age matched with

inactive SLE under maintenance therapy with only corticosteroid who were at regular follow-

up visit and 15 sex- and age-matched healthy subjects as a disease control from outpatient

clinic of Internal medicine and dermatology departments, Tanta university hospital, Egypt.

All patients fulfilled the American College of Rheumatology (ACR) 1997 revised criteria of

SLE 10. Disease activity index was assessed by using the ACR SLE disease activity index

(SLEDAI) 11.

4

For all participants, history taking, clinical examination, ANA, anti-double-stranded DNA,

and anti-Sm antibodies C3 and C4 were assessed. We quantified Toll-like receptor 9 mRNA

expression for SLE patients (active and nonactive) and healthy subjects.

Patients with active SLE (25 patients) received only oral steroid therapy (dose 1 – 2 mg/kg)

for 6 weeks. Post therapy, they were classified into steroid sensitive and steroid resistant

according to their post-therapy SLEDAI score. Decrease of SLEDAI score ≥4 from their

pretreatment score were considered as steroid sensitive. For both steroid sensitive and steroid

resistant, laboratory data and Toll-like receptor 9 mRNA expression were re-assessed.

The protocol of the study was approved by the research committee of the Faculty of

Medicine, Tanta university hospital. Informed written consent prior to participation in the

study was obtained from all patients and healthy subjects.

Sample collection

Blood samples.

Blood samples (7 ml) were obtained from patients and controls; 3 ml of the sample was

collected to an EDTA tube for performing DNA extraction and 2 ml in another tube to

perform complete blood picture. The rest of the samples were centrifuged at 3000 × g for 10

min at 4ºC, after that serum was removed and used for determination of C3 and C4 by

nephelometry (Behring GmbH, Marburg, Germany) 12, ANA, anti-DNA, and anti-Sm

antibodies were determined by enzyme-linked immunosorbent assays (Orgentec Diagnostika

GmbH, Mainz, Germany). Urine analysis, 24-h urinary protein excretion (UPE) 13, and

creatinine clearance 14 were determined.

RNA extraction

5

Extraction of RNA was done from blood samples by Qiagen RNeasy Mini Kit according to

the protocol supplied by the manufacturer. One volume of 70% ethanol was then added to the

cleared lysate and mixed immediately, then transferred to Qiagen RNeasy spin column placed

in a 2-ml collection tube and proceeded according to the manufacturer's instructions. RNA

was eluted and its concentration was measured spectrophotometrically (280). Five hundred

nanogram of total RNA was used for first-strand cDNA synthesis primed with 50 pmol of

random primer (Roche, Mannheim, Germany) and 0.5 µl (200 U/µl) reverse transcriptase

Superscript™ II-RT (Invitrogen, Karlsruhe, Germany). PCR cycling condition was performed

according to the manufacturer's instructions.

Reverse transcriptase – PCR 15.

One microliter of the reverse transcriptase reaction mixture was added to a 20-µl reaction

mixture of the QuantiTect SYBR-Green PCR kit (Qiagen) and 0.5 µM from the specific

primer pair for human TLR9-forward, 5-TAG AGA CTT CAG GCC CAA CTG-3, and

TLR9-reverse, 5-TGC ACG GTC ACC AGG TTG T-3. Primers for GAPDH are GAPDH-

forward ATG GCT ATG ATG GAG GTC CAG and GAPDH-reverse, TTG TCC TGC ATC

TGC TTC AGC. A negative control containing the complete Master Mix without a DNA

template was included. Samples of cDNA were assayed on the step one Real Time PCR

System (Applied Biosystems). Initial denaturation at 95˚C for 900 sec was followed by 45

cycles with denaturation at 95˚C for 15 sec, annealing at 64˚C for 20 sec and extension at

72˚C for 20 sec. The fluorescence intensity of SYBR-Green, specifically incorporated in the

double-stranded DNA amplicon reflecting the amount of formed PCR product was read after

each extension step at 72˚C. RNA amounts were determined with the Applied Biosystems

software in a mode relative to GAPDH gene (Figure 1).

6

Statistical analysis

Data were analyzed with the GraphPad InStat. Descriptive data were given as mean ±

standard deviation (SD). Comparison between the two groups was performed with t-test, but

among different groups, ANOVA was preformed followed by Tukey–Kramer Multiple

comparisons test. A spearman rank correlation was performed to determine the associations

between the studied parameters and TLR9. A p value less than 0.05 was considered

statistically significant.

Results

This study was performed on 25 patients with newly diagnosed active SLE (F/M was 20:5)

with SLEDAI score >4 and 15 patients with inactive active SLE (F/M was12:3) with

SLEDAI score ≤4. Fifteen sex- and age-matched healthy subjects (F/M was12:3) were also

included as control group. Table I shows clinical characteristics of SLE patients.

The SLEDAI score was significantly higher in active group versus inactive group (p < 0.001).

Corticosteroid maintenance dose ranged from 5 to 20 mg/day in inactive group while it was

50 – 80 mg/day in active group with (p < 0.001). ANA and anti-DNA were significantly

higher in inactive and active groups as compared with the control group (p < 0.001). The

serum concentration of C3 and C4 were significantly decreased in active group as compared

with the inactive and control groups (p < 0.001) while there was no significant difference

between inactive and control groups (p > 0.05). Anti-SM and TLR9 expression were

significantly higher in the active group as compared with the inactive and control groups (p <

0.001) while there was no significant difference between inactive and control groups (p >

0.05) (Table II).

7

Based on retrograde analysis, there was no significant difference between steroid-sensitive

and steroid-resistant group regarding corticosteroid dose received (p > 0.05). Table III shows

comparison of clinical and biochemical parameters before and after treatment within steroid-

sensitive and steroid-resistant groups separately. SLEDAI score, ANA, and anti-DNA were

significantly decreased in steroid-sensitive group after treatment while they were significantly

increased steroid-resistant group after treatment. The serum concentration of C3 and C4 was

significantly increased in steroid-sensitive group after treatment (p < 0.001) while there was

no significant difference of their values after treatment in steroid-resistant group (p > 0.05).

There was no significant difference of TLR9 expression value within the same group after

treatment versus before treatment (p > 0.05).

Table IV shows that SLEDAI score, and anti-DNA were significantly higher in steroid-

resistant group when compared with steroid-sensitive group either before or after treatment (p

< 0.001). Regarding ANA, there was no significant difference between steroid-sensitive and

steroid-resistant group before treatment (p > 0.05), while after treatment, ANA was

significantly higher in steroid-resistant group when compared with steroid-sensitive group (p

< 0.001). Anti-SM shows no significant difference between steroid-sensitive and steroid-

resistant groups either before or after treatment (p > 0.05). The serum concentration of C3

and C4 were significantly higher in steroid-sensitive group when compared with steroid-

resistant group either before treatment or after treatment (p < 0.001).

TLR9 expression was significantly higher in steroid-resistant versus steroid-sensitive group

either before treatment or after treatment (p < 0.001). There was a significant positive

correlation between TLR9 expression and SLEDAI score and Anti-DNA (IU/mL) (p < 0.05).

Also, there was a significant negative correlation between TLR9 expression and serum

8

concentration of C3 and C4 (p < 0.05) while there was no correlation between TLR9

expression and anti-SM (p > 0.05). (Table V)

Using the ROC curve, TLR9 expression level >6.54 was estimated to be the cut-off value for

detection of non-responder in active SLE patients with 0.738% accuracy, 85.7 negative

predictive value, 66.7% positive predictive value, 80.0 sensitivity, and 75.0% specificity

(Table VI).

Discussion

Over the last several years, new emphasis has been placed on the role of the innate immune

system in amplification of autoimmune response and exacerbation of SLE. Toll-like receptors

have been recognized as essential component in innate immunity and promote the generation

of autoantibodies against nuclear components.

In the present study, TLR9 was significantly higher in patients with active SLE compared

with patients with inactive SLE and controls and it correlated positively with ANA, anti-

DNA, and SLEDAI score. Our results support the previously published studies that raise the

possibility of TLR9 having a role in the pathogenesis of SLE. A study by Nakano et al. 16

demonstrated that higher expression of TLR9 on peripheral blood B cells from patients with

active SLE was significantly correlated with SLEDAI and induced anti-dsDNA antibody and

IL-10 through ligation of CpG to TLR9 on B cells. Using cytoplasmic staining and RT-PCR,

they confirmed that the expression of TLR9 was increased on B cells from patients with

active SLE, and this was correlated with disease activity. Furthermore, deficiencies of TLR9

suppress the production of anti-RNA or anti-DNA autoantibodies in murine models of SLE

17.

9

TLR induced the expression of co-stimulatory molecules, as represented by CD80 and CD86,

and the expression of TLR9 was correlated with that of CD86 on B cells. Since the

expression of CD86 on B cells is commonly increased in active SLE, it is possible that the

increased expression of TLR9 induces the initial activation of B cells. Thus, TLR9 expression

on B cells plays a role in SLE pathogenesis through the induction of anti-dsDNA antibody.

Moreover, methylation of DNA is decreased in SLE patients, suggesting that the interaction

between CpG and TLR9 plays a pivotal role in anti-dsDNA antibody production 18

Several studies have demonstrated that anti-nucleic acid immune complexes, isolated from

the sera of SLE patients, stimulate plasmacytoid dendritic cells (pDCs) to produce IFN-α 19,

20. Mean et al. 21 have reported that DNA-containing immune complexes, but not protein-

containing immune complexes, stimulate pDCs to produce cytokines, including IFN-α, TNF,

and IL-8. This process is thought to be mediated by a cooperative interaction between TLR9

and FcγRIIa, supporting the “dual receptor paradigm” in promoting activation of the innate

immune system. Barrat et al. 22 showed that mammalian RNA and DNA, in the form of

immune complexes, are potent self-antigens for TLR7 and TLR9, respectively, and induce

IFN-a production by plasmacytoid dendritic cells.

In the present study, there was a significant positive correlation between TLR9 expression

and SLEDAI score and anti-DNA. Also there was a significant negative correlation between

TLR9 expression and serum concentration of C3 and C4. Recently, two study groups

examined the expression of TLRs in peripheral blood mononuclear cells (PBMCs) from SLE

patients by flow cytometry 6, 23. It has been demonstrated that the proportion of plasma cells

and memory B cells expressing TLR9 was increased in active SLE patients and correlated

with the presence of anti-dsDNA antibodies in their patients, which is in accordance with our

finding 6. In fact, patients with active SLE have increased expression of TLR9 in peripheral

10

blood memory and plasma B lymphocytes, suggesting that endogenous nucleic acids released

during apoptosis may stimulate B lymphocytes via TLR9 and contribute to SLE pathogenesis

24. In contrast to our finding, Migita et al. 23 also showed no significant correlation between

TLR9 expression levels and clinical markers such as SLEDAI scores and anti-dsDNA

antibodies in their patients. In experimental studies of murine lupus, there were also opposite

findings regarding the role of TLR9 in anti-DNA antibody formation 25,26. This discrepancy

might be due to the different genetic background of mice used in these studies.

To the best of our knowledge, our study is the first one evaluating the relation between TLR9

expression and response to oral glucocorticoids therapy in active human SLE patients.

In our study, 15 patients with active SLE were steroid sensitive with significant post-therapy

decrease in their SLEDAI score, while the other 10 patients were steroid resistant with

significant post-therapy increase in their SLEDAI score. By retrograde analysis of our

patients clinical and laboratory data before therapy, SLEDAI score and anti-DNA were

significantly higher, C3 and C4 were significantly lower in steroid-resistant group when

compared with steroid sensitive. TLR9 expression was significantly higher in steroid resistant

versus steroid sensitive either before treatment or after treatment. TLR9 expression did not

change significantly either in steroid-sensitive or steroid-resistant group upon treatment.

Thus, these data suggest that assessment of TLR9 expression can be used in predicting

glucocorticoid treatment response in active SLE patients.

Glucocorticoids induce apoptosis in many cell types, including PDCs, where TLR signaling

confers partial protection 7. Freshly isolated PDCs from healthy donors stimulated with

TLR9 ligands were protected from glucocorticoid-induced cell death. This dose-dependent

protection correlated with the production of IFN-α by PDCs supporting our data at a single

cell level 27. Blocking this pathway with IRS 954 (bifunctional TLR9 inhibitor called

11

immunoregulatory sequence) restored glucocorticoid sensitivity to PDCs in vitro although

IRS 954 itself was not cytotoxic 22. Moreover, signaling through TLR9 protects human

PDCs from glucocorticoid-induced cell death. Taken together, the human data may be

interpreted as being consistent with the experimental mouse studies and support the concept

that TLR9 activation by DNA-containing immune complexes is crucial to SLE pathogenesis

28.

Guiducci et al. 29 reported that in vitro and in vivo stimulation of PDCs through TLR9 can

account for the reduced activity of glucocorticoids to inhibit the IFN pathway in SLE patients

and in two lupus-prone mouse strains. The triggering of PDCs through TLR9 by nucleic acid-

containing immune complexes or by synthetic ligands activates the NF-κB pathway essential

for PDC survival. They added that glucocorticoids do not affect NF-κB activation in PDCs,

preventing glucocorticoid induction of PDC death and the consequent reduction of systemic

IFN-α level. These findings unveil a new role for self nucleic acid recognition by TLRs and

indicate that inhibitors of TLR9 signaling could prove to be effective corticosteroid-sparing

drugs. In experimental study 30, TLR9 activation with CpG-ISS (CPG immunostimulatory

sequence) afforded significant protection from glucocorticoid-induced cell death to

conventional and PDCs in both spleen and blood. Circulating PDCs are significantly more

susceptible to glucocorticoid-induced cell death than TLR-activated PDCs in vivo. In

addition, treatment of lupus-prone mice with a dual inhibitor of TLR9 leads to the reduction

of autoantibody production and amelioration of disease symptoms. Although the direct TLR

antagonists have not been studied in human patients, inhibitors of IFN-α, a primary

downstream effector of TLR signaling and important disease mediator in SLE, have been

developed and are currently undergoing clinical trials 31.

12

As a consequence, the accumulation of evidence pointing toward TLRs in autoimmunity has

opened the door for potential therapeutic interventions directed toward the modulation of

Toll-like receptors and their signaling pathway. Since there are several proteins involved in

TLR signaling, there are a number of targets that may be utilized for potential drugs. Some of

the possibilities include development of TLR antagonists, inhibitors of downstream signaling

events, activation of natural inhibitory molecules, or blockade of the effector molecules

produced 32. Although there has not been a new drug approved for the treatment of lupus in

many years, current investigation regarding the targeting of TLRs and their downstream

effectors is showing some promise and warrants high expectations.

Conclusion:

TLR9 may play a role in the pathogenesis of SLE and correlates with the disease activity.

Corticosteroids have no effect on TLR9 expression and this can explain lack of corticosteroid

response in some of SLE patients. TLR9 expression can be used in predicting glucocorticoid

treatment response in active SLE patients. New modalities of treatment targeting TLR9

expression and antagonizing their effect will be of value in SLE steroid-resistant patients.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References <journal>1. Mok CC, Lau CS. Pathogenesis of systemic lupus erythematosus. J Clin Pathol.

2003;56:481–90. <journal>2. Christensen SR, Shlomchik MJ. Regulation of lupus-related autoantibody production

and clinical disease by Toll-like receptors. Semin Immunol. 2007;19:11–23. <journal>3. Horton CG, Pan ZJ, Farris AD. Targeting Toll-Like receptors for treatment of SLE.

Mediators Inflamm. 2010;1-9. <journal>4. Heit A, Huster KM, Schmitz F, Schiemann M, Busch DH, Wagner H. CpG-DNA aided

cross-priming by cross presenting B cells. J Immunol. 2004;172:1501–7. <journal>5. Leadbetter EA, Rifkin IR, Hohlbaum AM, et al. Chromatin-IgG complexes activate B

13

cells by dual engagement of IgM and Toll-like receptors. Nature. 2004;416:603–7. <journal>6. Papadimitraki ED, Choulaki C, Koutala E, et al. Expansion of toll like receptor 9 -

expressing B cells in active systemic lupus erythematosus: implications for the induction

and maintenance of the autoimmune process. Arthritis Rheum. 2006;54:3601–11. <journal>7. Chatham WW, Kimberly RP. Treatment of lupus with corticosteroids. Lupus.

2001;10:140–147. <journal>8. Chaussabel D, Quinne C, Shen J, et al. A modular analysis framework for blood

genomics studies:application to systemic lupus erythematosus. Immunity. 2008;29:150–164.

<journal>9. Barrat FJ, et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005;202:1131–1139.

<journal>10. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725-1734.

<journal>11. Griffiths B, Mosca M, Gordon C. Assessment of patients with systemic lupus erythematosus and the use of lupus disease activity indices. Best Pract Res Clin Rheumatol. 2005;19:685–708.

<journal>12. Virella G. Nephelometric techniques in immunodiagnosis. Clin Immunol Newsl. 1980;1:1–16.

<journal>13.Viberti GC, Pickup JC, Jarrett RJ, Keen H. Effect of control of blood glucose on urinary excretion of albumin and beta 2 microglobulin in insulin- dependent diabetes. N Engl J Med. 1979;300:638–41.

<journal>14. Bartels H, Bohmer M. Micro-detremination of creatinine. Clin Chim Acta. 1971;32:81-5.

<journal>15. Komatsuda A, Wakui H, Iwamoto K, et al. Up-regulated expression of Toll-like receptors mRNAs in peripheral blood mononuclear cells from patients with systemic lupus

erythematosus. Clin Exp Immunol. 2008;152:482–487. <journal>16. Nakano S, Morimoto J, Suzuki, K, Nozawa H, Amano Y: Tokano Y. Role of

pathogenic auto- antibody production by Toll-like receptor 9 of B cells in active systemic

lupus erythematosus. Rheumatology. 2008;47:145–149. <journal>17. Ehlers M, Fukuyama H, McGaha TL, Aderem A, Ravetch JV. TLR9/MyD88 signaling

is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE. J Exp Med. 2006;203:553–561.

<journal>18. Sekigawa I, Okada M, Ogasawara H, Kaneko H, Hishikawa T, Hashimoto H. DNA methylation in systemic lupus erythematosus Lupus. 2003;12:79–85.

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Table I. Clinical characteristics of systemic lupus erythematosus (SLE) patients. SLE group (40) Variables Number %

Arthritis 24 60 Serositis 10 25 Vasculitis 11 27.5 Renal involvement 7 17.5 Hematological manifestation 17 42.5 Fever 23 57.5 Mucosal ulcer 15 37.5

Table II. Comparison between all studied groups with regard to the clinical and biochemical parameters

SLE (40) ANOVA Tukey's test Variable Control

15 Inactive 15

Active (25)

F p-value

I&II I&III II&III

Mean

3.50 21.15 SLEDAI Score

SD

1.83 4.58

13.76t

< 0.001

Mean 11.25 60.55

Corticosteroid dose

SD

2.55 5.33

33.472t

< 0.001

Mean

6.43 21.89 25.66 ANA (IU/ml)

SD 3.35 2.61 268.1

< 0.001

<0.001

<0.001

<0.001

15

Mean

1.58 81.70 Anti-DNA (IU/ml)

SD 5.70 36.10 9.02 171.6

< 0.001

<0.001

<0.001

<0.001

Mean 10.55 12.55 29.84

Anti-SM (IU/ml)

SD 1.53 1.21 3.66 7.8 0.001

0.949

<0.001

<0.001

Mean

146.30 138.81 58.23 C3 (mg/dl)

SD 27.29 7.32 6.62 208.9

< 0.001

0.393

<0.001

<0.001

Mean

35.22 32.56 9.10 C4 (mg/dl)

SD 3.97 2.23 1.85 595.5

< 0.001

0.26 <0.001

<0.001

Mean

1.61 1.88 6.41 TLR9 expression

SD 0.74 1.00 0.65 250.5

< 0.001

0.597

<0.001

<.001

Table III. The clinical and biochemical parameters before and after treatment within the same group.

Before After Mea

n SD Mean SD

% of change

Paired t-test

Steroid sensitive

18.4 3.7 10.6 2.65

–42.391 0.010 SLEDAI Score

Steroid resistant

25.5 1.3 29.3 1.88

14.902 0.031

Steroid sensitive

25.3 2.8 19.2 3.02

–24.111 0.022 ANA (IU/ml)

Steroid resistant

26.3 2.3 28.6 2.12

8.745 0.039

Steroid sensitive

76.7 6.7 57.3 5.55

–25.293 0.000 Anti-DNA (IU/ml) Steroid

resistant 89.8 5.9

105.6

4.66

17.595 0.000

Steroid sensitive

23.6 3.54

22.7 2.55

–3.814 0.512 Anti-SM (IU/ml)

Steroid resistant

25.66

3.55

24.52

4.12

–4.443 0.772

Steroid sensitive

61.8 5.3 93.2 4.55

50.809 0.000 C3 (mg/dl)

Steroid resistant

52.5 3.9 49.72

2.78

–5.295 0.128

Steroid sensitive

10.3 1.1 21 2.05

103.883 0.000 C4 (mg/dl)

Steroid resistant

7.2 1.1 6.93 1.82

–3.750 0.243

Steroid sensitive

6.03 0.49

5.9 0.56

–2.156 0.096 TLR9 expression

Steroid resistant

7.01 0.33

7.37 0.46

5.136 0.722

16

Table IV. Comparison between steroid-sensitive and steroid-resistant groups with regard to the clinical and biochemical parameters before and after treatment.

Steroid sensitive Steroid resistant T-test

Mean SD Mean SD t p-value Before 18.4 3.7 25.5 1.3 6.882 0.000

SLEDAI Score After 10.60 2.65 29.30 1.88 20.694 0.000 Before 25.3 2.8 26.3 2.3 0.977 0.338

ANA (IU/mL) After 19.20 3.02 28.6 2.12 7.609 0.000 Before 76.7 6.7 89.8 5.9 5.155 0.000

Anti-DNA (IU/mL) After 57.30 5.55 105.60 4.66 23.535 0.000 Before 23.6 3.54 25.66 3.55 1.423 0.168

Anti-SM (IU/mL) After 22.70 2.55 24.52 4.12 1.242 0.227 Before 61.8 5.3 52.5 3.9 5.062 0.000

C3 (mg/dl) After 93.20 4.55 49.72 2.78 26.943 0.000 Before 10.3 1.1 7.2 1.1 6.903 0.000

C4 (mg/dl) After 21.00 2.05 6.93 1.82 17.555 0.000 Before 6.03 0.49 7.01 0.33 5.997 0.000

TLR9 expression After 5.90 0.56 7.37 0.46 7.180 0.000

Table V. Correlation between the TLR9 expression and other variables in all systemic lupus

erythematosus (SLE) patients.

TLR9 expression Variables

r p-value

Score 0.625 0.021

Corticosteroid dose 0.125 0.890

ANA (IU/ml) 0.241 0.688

Anti-DNA (IU/ml) 0.651 0.021

Anti-SM (IU/ml) 0.116 0.900

C3 (mg/dl) –0.771 0.001

C4 (mg/dl) –0.702 0.005

Table VI. ROC curve for detection of cut-off value of TLR9 expression in detecting non-

responder among active systemic lupus erythematosus (SLE) patients.

ROC curve Cut-off Sens. Spec. PPV NPV Accuracy >6.54 80.0 75.0 66.7 85.7 0.738 Figure 1. Amplification plot of TLR9 and GAPDH showing the log of the change in the fluorescence plotted against cycle number.