| Background Systemic lupus erythematosus (SLE) is prototype of autoimmune disease characterized by the disfunctions of T cell and B cell, the generation of pathogenic autoantibodies such as anti-DNA autoantibodies, and the occurrence of immune complexes (IC). And the deposit of autoantibodies and ICs can lead to inflammation and injury of multiorgans. SLE, the clinic manifestations of which are variable including glomerulonephritis, dermatitis, vasculitis, epileptic seizure, arthritis and so on, is a type of complex diseases and also regarded as the multifactorial disease. Up to nowadays, although there have been a lot of hypothesises about the etiology and pathogenesis of SLE such as the immunology theory, the genetics theory and theory of the interaction between environment and genetics,the exact development mechanisms of SLE are elusive. In present years, more attention has been paid to the role of Toll-like receptors family in the pathogenesis of SLE,especially toll-like receptor 9 (TLR9).TLR9, a hot point in immunity, whose ligand being hypomethylated CPG-DNA, plays a controversial role in lupus, whose protective or promoting effects were both reported in human and murine lupus, in vitro or in vivo, suggesting the influences of different genera, genetic backgrounds and environment conditions on approaching its role.In SLE, there is the imbalance of Th1/Th2 immunity, and T-box transcription factor T-bet (Tbx21) has emerged as a key regulator of type 1-like immunity. Recently, T-bet also has been considered as a bridage between the innate immunity and the acquired immunity as the TLR9. And some literature reported TLR9 is unique to the production of the anti-DNA autoantibodies, and the absence of TLR9 signaling resulted in less T-bet production.T-bet,playing crucial roles in the establishment and/or maintenance of effector cell fates in T and B lymphocytes,regulates T-independent IgG2a class switching in murine models. So, T-bet may be involved in the processes between the initiation of TLR9 activation and the antibodies'production. And the TLR9 and T-bet may have close relationships with the production of pathogenic antibodies and the pathogenesis of SLE, in particular, the TLR9 and T-bet may correlate with each other.To clarify the paradox of TLR9 and the pathway of TLR9-T-bet-antibody in the pathogenesis of SLE, we investigated the intracellular expressions of TLR9 and T-bet in B and T cells in peripheral blood samples from newly diagnosed, untreated patients with SLE and healthy subjects by flow cytometry (FCM).Objectives To compare the expressions/coexpressions of TLR9/T-bet in the B/T cells between the SLE patients and healthy controls.To analyze the relationships between the expressions of TLR9/T-bet and the coexpressions of TLR9 and T-bet in the B/T cells and SLE disease activity,clinical laboratory data,clinical manifestations, and the relationship between the TLR9 and the T-bet for finding the role of TLR9-T-bet pathway in the SLE pathogenesis.Methods SLE patients were selected from two provincial hospitals, and normal healthy volunteers were recruited as controls.General epidemiological information, the data of clinical manifestations and clinical laboratory indexes were collected by self-designed questionnaire according to the systemic lupus erythematosus disease activity index(SLEDAI) and the diagnostic and classification criteria. The three-color FCM system was used to detect the intracellular expressions of TLR9 and T-bet in B and T cells in peripheral blood samples from 35 newly diagnosed, untreated patients with SLE and 16 healthy subjects with FITC-conjugated anti-human T-bet, PE-conjugated anti-human TLR9, PE-Cy5-conjugated anti-human CD3-, PE-Cy5-conjugated anti-human CD19 antibodies.Spss was used to analyze the differences and relationships between the data.Results TLR9 ( p =0.000), T-bet ( p =0.001) were expressed on a statistically significantly elevated percentage of B cells in SLE patients (mean±SD 53.94±17.95% and 22.77±16.80%, respectively) when compared with healthy controls (mean±SD 29.40±10.54% and 11.46±6.56%, respectively). And the proportion of T cells expressing TLR9 was statistically significantly higher ( p =0.001) in patients (mean±SD 49.33±23.30%) with SLE than that in healthy subjects (mean±SD 29.18±14.78%) A statistically significant negative correlation between the proportion of B cells expressing TLR9 and SLEDAI score was found ( p =0.020, r =?0.392 by Pearson's correlation). And there is no other statistically significant association was found between SLEDAI score and the expressions of TLR9/T-bet or coexpressions of TLR9 and T-bet in circulating B/T cells.The serum levels of anti-C1q antibody showed a statistically significant correlation with the proportion of B cells expressing T-bet (Pearson's correlation p =0.011, r =0.424).The correlations between the serum levels of IgA antibody and the percentage of B cells expressing TLR9, B cells co-expressing TLR9 and T-bet, and B cells expressing T-bet were statistically significant (Pearson's correlation p =0.000, r =0.742, p =0.014, r =0.422, p =0.006, r =0.458, respectively).Besides the percentages of the cells expressing or coexpressing TLR9/T-bet, the expressions of TLR9/T-bet or coexpressions of TLR9 and T-bet per B /T cell, expressed as the mean fluorescence intensity (MFI) were also determined. And some statistically significant correlations were found between MFIs and laboratory data. The serum levels of anti-dsDNA antibody significantly inversely correlated with the MFI of B cells co-expressing T-bet and TLR9 (Spearman's correlation p < 0.000, rs =?0.582), but did not correlate with the percentage of B cells co-expressing T-bet and TLR9 significantly. The serum levels of the IgM antibody statistically significantly correlated with the MFI of T cells co-expressing T-bet and TLR9 (Pearson's correlation p =0.001, r =0.540), but not significantly with the percentage of T cells co-expressing T-bet and TLR9 .No data suggest any statistically significant association of the expressions/coexpressions of TLR9/T-bet in circulating B/T cells with the serum levels of anti-nuclear antibodies (ANA), anti-nucleosome antibody, CRP, C3, C4, and ESR, etc.The inter-related associations among the percentages of CD19+/CD3+ cells expressing TLR9/T-bet and/or the MFIs of CD19+/CD3+ cells expressing TLR9/T-bet in patients with SLE were all positively statistically significantThe followings are about the relationships between the expressions of TLR9/T-bet in the circulating B/T cells and the clinical manifestations of SLE. The percentages of T cells expressing TLR9 in SLE patients with serositis, vasculitis or pleurisy were significantly lower than those without, being 33.90±10.10%, 24.60±3.42% and 33.00±8.98% in the former, and 51.32±23.85%, 51.65±23.02% and 52.05±23.91% in the latter, respectively. The percentages of T cells expressing T-bet in SLE patients with hematologic disorder is significantly lower than those without,being 25.38±13.93%,35.82±16.08%, respectively.The percentages of B cells expressing TLR9 in SLE patients with myositis, cylindruria, pyuria or Leukopenia are significantly lower than those without, being 37.45±0.64%,36.52±12.79%,43.27±19.22% and 48.26±16.76% in the former and 54.94±18.01%,56.84±17.15%,57.63±16.28% and 60.68±17.44% in the latter, respectively. But the percentages of B cells expressing T-bet in SLE patients with tuberculosis is significantly higer than those without, being 49.55±27.65% and 21.15±15.12%, respectively.The MFIs of T cells expressing T-bet in SLE patients with myositis is significantly higher than those without, being 21.65±5.44 in the former and 16.03±3.64 in the latter,respectively. The MFIs of B cells expressing TLR9 in SLE patients with thrombocytopenia is significantly lower, but with renal disorder is higher than those withour, being 10.93±1.64 and 12.46±4.42 in the former and 17.04±9.91 and 19.48±11.74% in the latter, respectively. The MFIs of B cells expressing T-bet in SLE patients with hematuria and Sjogren Syndrome are significantly higher than those without,being 20.75±5.95 and 25.33±8.60% in the former and 17.13±4.20 and 17.83±4.40 in the latter, respectively..The percentage of B cells co-expressing TLR9 and T-bet in SLE patients with hematologic disorder is significantly lower, and with tuberculosis is significantly higher than that without,being 10.16±5.42% and 44.65±32.60% in the former and 18.88±15.07% and 12.56±7.49% in the latter, respectively.. The percentages of T cells co-expressing TLR9 and T-bet in SLE patients with hematologic disorder, thrombocytopenia and leucopenia are lower than those without,being 11.20±7.04%, 10.16±6.63% and 12.70±7.93% in the former and 24.8±15.47%, 20.50±14.47% and 23.95±16.48 %in the latter,respectively.Conclusions Generally speaking, expressions of TLR9 and T-bet were increased in patients with SLE compared with controls and TLR9 may have a role to play in protecting against lupus. And the increase of the co-expression of TLR9 and T-bet may be of benefit to the protective antibodies'production and the pathogenic antibodies'decline. Expressions of TLR9 and T-bet have negative relationships with onsets of most SLE clinical characteristics in the SLE classification and SLEDAI scales,but positive relationships with hematuria in renal disorder and complications.of SLE and the co-expression of TLR9 and T-bet could be regarded as a good sign for lupus demission and/or treatment.
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