| Background: Nucleic acid is released during the pathogen infection or in the event of tissue damage.Normally,Toll-like receptors(TLRs)in vivo recognize nucleic acids of invasive pathogenic microorganisms and activate the innate immune system to protect the body from pathogenic microbes.However,in the case of pathology,the body's persistent natural immune response to its own nucleic acid can lead to a variety of autoimmune diseases.Toll like receptor 9(TLR9)mainly identify double-stranded DNA and could be active by immune complex including self double-stranded DNA,resulting in Systemic lupus erythematosus(SLE)disease.Additionally,previous study reported that TLR9 expression correlated with C3 in SLE patients.And treatment with TLR9 agonists could lead to thrombocytopenia(TCP).However,it remains unclear how TLR9 involved in TCP-SLE.Although there are many studies on TLR9 in SLE,the role of TLR9 in SLE remains unclear.It is sure that TLR9 plays a key role in SLE.Therefore,in-depth study of the clinical value of TLR9 and the mechanism of TLR9-related signaling pathway in systemic lupus erythematosus and its subtypes is a profoundly research.Objective: To investigate the expression of TLR9 / TGF-?1 / PDGF-B and TLR9 / TGF-?1 / C3 in SLE and TCP-SLE patients,respectivity.And explore the clinical value of TLR9 expression in SLE patients,especially prognostic value of TLR9 within 2 years for SLE patients.Methods: SLE patients(n=112),subtype TCP-SLE patients(n=38)and age-and sexmatched healthy controls were enrolled.The expression of TLR9,TGF-?1,PDGF-B and C3 m RNA in whole blood cells were detected by q RT-PCR.The plasma protein levels of TLR9,TGF-?1 and PDGF-B were detected by ELISA.SLE patients were followed-up for recording prognosis within 2 years.Results: 1.The levels of TLR9,TGF-?1 and PDGF-B in peripheral blood of SLE patients were higher than those of healthy controls(HCs): the m RNA level of TLR9 in peripheral blood of SLE patients was significantly higher than that of HCs(p = 0.0048),TGF?1 and PDGF-B protein levels were significantly higher than those in healthy controls(p <0.0001 and p = 0.0084).After therapy,the levels of TGF-?1 and PDGF-B m RNA were significantly decreased(p <0.0001 and p = 0.0255).2.There was significant correlation between TLR9,TGF-?1 and PDGF-B in HCs and SLE patients: the protein level of PDGF-B was correlated with the level of TGF-?1 protein both in peripheral blood of HCs(p <0.0001,r = 0.61)and SLE(P <0.0001,r = 0.68).Meanwhile,the m RNA levels of TGF-?1 correlated with PDGF-B both in HCs and SLE(p = 0.0268,r = 0.33;SLE patients p <0.0001,r = 0.61).Furthermore,significant correlation between m RNA levels of TLR9 and TGF-?1(p = 0.0003,r = 0.51)and great correlation between m RNA levels of TLR9 and PDGF-B(p = 0.0018,r = 0.45)were detected in healthy controls.Moreover,it was found that m RNA levels of TLR9 can be correlated with TGF-?1(p <0.0001,r = 0.52)or PDGF-B(p <0.0001,r = 0.45)in SLE patients very well.3.The levels of MCP-1(p = 0.0039,Figure 3A),ISG15(p = 0.0004)and IFN?(p = 0.0073)increase significantly in the peripheral blood of SLE patients comparing to that of healthy controls.Unlike TGF-?1 and PDGF-B,no significant correlation was found between TLR9 and MCP-1(p = 0.2544,r = 0.1091),TLR9 and ISG15(p = 0.9722,r =-0.0072)or TLR9 and IFN?(p = 0.1247,r =-0.3549).4.Cp G significantly up-regulate the m RNA levels of TGF-?1 and PDGF-B in healthy controls(p = 0.0210;p = 0.0093)and SLE patients(p = 0.0005;p = 0.0005).Cp G can induce much higher levels of TGF-?1(p = 0.0485)and PDGF-B(p = 0.0037)in SLE patients than healthy controls.Similar results were also found in isolated monocytes from SLE patients and healthy controls.5.TGF-?1 increased protein levels(p = 0.0156)and m RNA levels(p = 0.0020)of PDGF-B significantly.On the other side,TGF-?1 antagonists,TGF-? RI/ALK inhibitor SB431542 and neutralizing anti-TGF-?1 monoclonal antibody 1D11 inhibited the production of PDGF-B significantly at both protein levels(p <0.05)and mRNA levels(p <0.05).Moreover,SB431542 significantly inhibited the PDGF-B production induced by Cp G(p <0.05).6.Supernatant of Cp G-stimulated primary cells from SLE patients can potently induce mesangial cell proliferation(p = 0.0171).By adding plasma to the culture medium of mesangial cells and measuring the cell-proliferation,it was found that plasma from SLE patients induced much higher proliferation rate than that from healthy controls(p = 0.0116).Anti-PDGF-B neutralizing antibody inhibited mesangial cell-proliferation induced by patients' plasma in a dose dependent manner(p <0.05).7.Significant correlations were found between the m RNA expressions of TGF-? and PDGF-B(p = 0.0092,r = 0.61),the m RNA expressions of TLR9 and TGF-?(p = 0.0003,r = 0.51)and the m RNA expressions of TLR9 and PDGF-B(p = 0.0052,r = 0.44).Positive correlations were also found between the protein levels of TGF-? and PDGF-B(p <0.0001,r = 0.64)in these patients.The protein levels of PDGF-B homodimer correlated with the levels of urine protein(p <0.0027,r = 0.49,Figure 7E)in SLE patients with LN.8.Increased gene expression of C3,TLR9 and TGF-?1 expressions in whole blood cells of SLE patients with TCP: Plasma C3 levels in lupus TCP patients were significantly lower than levels in SLE patients without TCP(p = 0.005).In contrast,the level of C3 m RNA expression in whole blood cells was significantly higher in lupus TCP patients compared with that in SLE patients without TCP(p < 0.01).Moreover,levels of TLR9 and TGF-?1 m RNA expression by whole blood cells from SLE patients with TCP were also significantly higher than those in SLE patients with TCP(p < 0.05 for TLR9 and TGF-?1).9.In SLE patients with TCP,TLR9 m RNA level in the group with high anti-ds DNA antibody titer was significantly higher than that in the low anti-ds DNA antibody titer group(p<0.05).Furthermore,the levels of plasma C3 negatively correlated with the SLEDAI scores and the titers of anti-ds DNA antibodies(r=-0.33,p=0.0411 for SLEDAI;r=-0.37,p=0.0207 for anti-ds DNA antibody titers).The levels of plasma C3 negatively correlated with the relative levels of C3 m RNA transcripts in whole blood cells(r =-0.41,p = 0.0113),but positively correlated with platelet counts in SLE patients with TCP(r = 0.37,p = 0.0211).In addition,the levels of TGF-?1 m RNA transcripts positively correlated with those of TLR9(r = 0.62,p < 0.0001)and C3(r = 0.45,p = 0.0043)in whole blood cells from SLE patients with TCP.10.SLEDAI scores of those patients responded to therapies were significantly reduced while blood platelet counts and the levels of plasma C3 were significantly elevated compared with those before treatments(p < 0.05 for SLEDAI and platelet counts,p < 0.01 for C3).Further analysis indicated that the levels of TLR9,TGF-?1 and C3 m RNA transcripts in whole blood cells from those patients responded to therapies were significantly lower than those before treatment(p < 0.05 for all).11.Treatment with Cp G significantly increased the levels of TGF-?1 in whole blood cells.We also found that stimulation with TGF-?1 significantly increased C3 expression;while treatment with SB431542,the TGF-? RI/ALK inhibitor,significantly reduced C3 expression in whole blood cells.Furthermore,treatment with Cp G to activate the TLR9 also increased significantly the levels of C3 expression in whole blood cells,which was completely abrogated by treatment with SB431542.12.TLR9 m RNA level was increased in the high-ds DNA Ab group compared with the low-ds DNA Ab and in the low-C3 level group compared with the normal-C3 level group(P=0.026 and P=0.001,respectively).There was no difference in TLR9 m RNA levels when comparing patients with renal involvement and those with no renal involvement(P=0.120).13.During the 2-year follow-up,52 patients(57.8%)experienced clinical remission and the remaining 38 patients(42.2%)were categorized into the poor prognosis group at different times.In univariate analyses,persistent proteinuria of >0.5 g per day(HR,3.81;95% CI,1.96-7.40),serum C3 level(HR,0.31;95% CI,0.09-0.98),SLEDAI(HR,1.05;95% CI,1.02-1.09),C-reactive protein(HR,1.006;95% CI,1.001-1.012)and high-TLR9 m RNA level(HR,3.60;95% CI,1.88-6.89)were predictive of poor prognosis in the 2-year follow-up period(Table 2).After adjusting for several factors,we observed that only persistent proteinuria(>0.5 g/day;HR,6.31;95% CI,2.86-13.95),C-reactive protein(HR,1.013;95% CI,1.007-1.019)and high-TLR9 m RNA level(HR 3.85;95% CI,1.93-7.68)were independent risk factors for poor prognosis,whereas treatment with more than one immunosuppressant for therapy(HR,0.37;95% CI,0.17-0.81)was an indicator of favorable prognosis.14.The median time of the SLE patients developing poor prognosis was 6 months after diagnosis(range,0-13.83 months).Kaplan-Meier survival curves(Figure 2)indicated that the 2-year poor prognosis-free probability was 66%(95% CI,54.2-77.8)in the low-TLR9 group,and 30%(95% CI,10.4-49.6)in the high-TLR9 group(HR,3.85;95% CI,1.93-7.68;P=0.000).15.There was significant increase in TLR9 m RNA levels in the poor prognosis group compared with the favorable prognosis group at baseline(P<0.0001).Compared with the levels at baseline,the TLR9 m RNA level decreased significantly after 2-years in the favorable prognosis group(P<0.0001),but remained high in the poor prognosis group(P=0.1192).Conclusions: 1.TLR9/TGF-?/PDGF-B pathway in human and can be excessively activated in SLE patients.High levels of PDGF-B may result in over-proliferation of mesangial cells in the kidney.2.Blood cells are source of extra-hepatic synthesis of C3 in TCP-SLE patients and this synthesis of C3 was up-regulated by TLR9 via induction of TGF-?1.Thus TLR9/TGF-?1/C3 pathway might be in operation mediating lupus thrombocytopenia.3.TLR9 is correlated with disease activity and may predict SLE outcome within a 2-year period.TLR9 may be a useful biomarker for predicting SLE prognosis. |
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