The Expression And Clinical Significance Of Toll-like Receptor 9 On Blood B Cell And Renal Tissue In Patients With Lupus Nephritis

[Background and objective]Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmunedisease characterized by overactivation of self-reactive T and B cells, production pathogenic autoantibody and immune complexes. Lupus nephritis (LN) is one of the most frequent and serious complications that occurs in this nonorgan-specific disease. Mechanism of LN remains to be unknown. In 10-25% of LN the kidney disorder progresses to end-stage renal failure, which is the crucial factor of prognosis in SLE.Toll-like receptors (TLRs) are an important family of receptors binding of the pathogen in innate immunity that recognize pathogen-associated molecular patterns (PAMP), then promote the activation of immunological effector molecules and product multiple pro-inflammatory cytokines to remove invading microbial pathogens. The recent discovery of the role of TLRs extends beyond microbial recognition that suggests a role for adaptive immune by binding their specific ligands including endogenous and exogenous molecules. This function is accomplished through coordinated up-regulation of major histocompatibility complex class II and costimulatory molecules, resulting in much more efficient antigen presentation with APC. Furthermore, TLR-induced secretion of type I IFNs, IL-6, IL-12 and tumor necrosis factor-a directs maturation and sublineage commitment of immune cells, such as CTL, B cell, that participate in the adaptive immune response. Therefore, TLRs bridge innate and adaptive immune responses and have focused attention on the autoimmune disease. TLR9, the member of TLR family, recognizes unmethylated CpG motifs in microbial DNA and immune complex that leads to activation of NF-κB,AP-1 through MyD88-dependent signaling pathways. After translocation into the nucleus NF-κB or AP-1 induces the expression of many pro-inflammatory genes finally products multiple cytokines and actives immune cells. TLR9 signaling participate B-cell survival, activation and autoantibody production and may play an important role in the pathogenesis of autoimmunity and inflammation. Thus some data support the hypothesis that CpG-DNA facilitates progression via TLR9 signaling in SLE and LN. Our purpose herein is to assess that the TLR9 singling involved in the pathogenesis of lupus disease activity and organ lesion, while perhaps indicates the potential role of infection-associated exacerbation of disease. Moreover, the further studies by TLR9 signaling may result in a novel way of management for patients with LN.In this study, the expressions of TLR9 were examined on the renal tissue and peripheral blood B lymphocyte of LN patients and healthy controls by immunohistochemistry and intracellular flow cytometry respectively. The correlations between TLR9 and clinical parameters were also analyzed, in order toinvestigate the mechanism and clinical significance of TLR9 in patients with LN.[Methods](1) Intracellular expression of TLR9 in peripheral blood CD19+ B lymphocyteswere analyzed in 39 LN patients and 18 healthy subjects by flow cytometry. According to the SLE Disease Activity Index (SLEDAI), LN patients were divided into two groups: the disease active group and the inactive group. The difference of intracellular TLR9 expression levels in three groups were compared and evaluated the correlations of it in B cells with clinical measures such as the SLEDAI, complement C3.(2) Renal expression of TLR9 was examined in renal biopsy specimens from 57 LN patients and 11 healthy controls by immunohistochemical method. According to the ISN/RPS revised pathologic classification of LN, the different TLR9 expression levels of each class of LN was compared. In addition, the renal histological changes and clinical parameters were analyzed as elucidated the relationship between expression of TLR9 and disease status.[Results](1) Compared with healthy subjects, levels of intracellular TLR9 expression of B lymphocytes were elevated in patients with LN, especially in those whose disease activity score were greater than 10 by SLEDAI (P<0.01). There was no significant difference between the inactive group and the healthy control (P>0.05). In addition, the increased expression of TLR9 in B cells was not significant difference in the presence or absence of moderate doses of corticosteroids or immunosuppressant.(2) The positive correlation was observed between TLR9 expression levels and clinical measure of the SLEDAI (P<0.01). But no correlation of TLR9 expression levels with complement C3 was found (P>0.05).(3) In kidneys of LN patients, TLR9 was expressed in infiltrating mononuclear cells in glomerular, interstitial, and perivascular cell infiltrates but not in those fields of healthy controls (P<0.05), while it accumulated in tubular cells both in LN patients and the controls. Moreover, the expression of TLR9 was more prominent in the kidneys with severe proliferative histopathology lesions. Compared with the ISN/RPS revised pathologic class II, class III and class V, the highest expression of TLR9 was observed in class IV(P<0.01).(4) The expression of TLR9 in glomeruli and renal interstitium in LN patients show a strong positive correlation with LN histopathology activity index (P<0.01). The positive relationship between TLR9 expression in glomeruli and glomerular cellular proliferation was found (P<0.01), as well as TLR9 expression in interstitium and the tubulointerstitial index(P<0.01).(5) Higher expression of TLR9 in glomeruli associated with decreased estimated glomerular filtration rate (P<0.05) and increased 24-h urinary protein (P<0.01). The similar correlation was observed between TLR9 expression in interstitium and eGFR (P<0.01), but no correlation with 24-h urinary protein (P>0.05). Neither glomeruli nor interstitium expressed TLR9 had correlation withcomplement C3 (P>0.05).[Conclusions](1) TLR9 expression up-regulated on peripheral blood B cells in LN patients,suggesting that TLR9 might be related to the abnormal B cell hyperactivity and the abnormal immune process in LN patients.(2) TLR9 expression increased on renal tissue of LN patients and the expression level correlated with severity of renal histological damage.(3) TLR9 levels on peripheral blood B cells and renal tissue may help in assessing disease activity, severity of renal damage and prognosis in LN patients.