Reduced expression of toll-like receptor 4 in B-chronic lymphocytic leukemia patients with autoimmune complications

Background: B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-like monoclonal B lymphocytes, defective apoptosis, progressive hypogammaglobulinemia with immunodeficiency, and high prevalence of autoimmune phenomena.Toll-like receptors (TLRs) are a pattern of type I integral membrane glycoproteins expressed on cells of the immune system and represent major agents of innate immunity and initiators of adaptive immunity. Aims: To evaluate TLR4 and TLR9 genes and protein expression in B-lymphocytes from B-CLL patients, and its relationship with stage, therapy, and known prognostic markers (IgVH region mutational status, cytogenetic alterations,CD38 and ZAP70). Further aim was to correlate TLR4 and 9 expression with infections, autoimmunity and disease progressionMethods: TLR4 and TLR9 gene expression was evaluated in total RNA of B cell from 95 B-CLL patients and controls (pool of 10 healthy donors) by TaqMan® Gene Expression Assays on the model 7300 real-time PCR system (Applied BioSystems-Life Technology, Foster City, CA, USA). The results of relative quantitation (RQ) assays were analyzed using SDS Software (Applied BioSystems-Life Technology). Surface TLR4 and TLR9 expression on B-lyphocytes was determined by standard flow cytometry. TLR4 expression was evaluated in cultured B cells, obtained with RosetteSep (purity >97%) either unstimulated or stimulated with 1mg/mL lypopolysaccharide (LPS). Culture supernatants were assayed for IL-10 and TGF-β production using commercially available ELISA kits. Results: Patients (41 females and 56 males, median age 74 years, range 41- 89) were 78.9% in Binet stage A, 11.6% in B, and 9.5% in C and were followed for a median of 27.6 months, range 19-44. Quantitative real-time PCR revealed that TLR4 gene expression was decreased (RQ=16.1+1.56) and TLR9increased (RQ=2725+165) in all B-CLL patients versus controls (RQ=100). Consistently, the percentage of CD19+ cells expressing TLR4 by cytofluorimetric analysis was lower (1.70+0.2% versus 3.93+0.68%, P=0.004) and TLR9 greater (5.5+0.6% versus 1.39+0.31%, P=0.04) in patients compared to controls. TLR4 reduction was more pronounced in advanced and multi-treated disease, and in patients with unmutated IgVH status and unfavorable cytogenetic abnormalities. Univariate Cox regression model showed that patients with reduced TLR4 gene expression had an increased risk of disease progression (HR 4.6, 95% CI 1.8-11.6; P=0.001) (Figure1, upper panel), suggesting that an impaired innate immunity identifies a subset of B-CLL patients with poor prognosis. Interestingly, patients with reduced TLR4 gene expression had an increased risk to develop autoimmune complications (P=0.02) (Figure1, lower panel). Finally, TGF-β production by B cells was increased in patients who thereafter developed autoimmune complications, compared with those that did not (1092+158 versus 793+18 pg/mL, mean+SE, P=0.07). Summary / Conclusion: Reduced expression of TLR4 in B-CLL patients correlates with an advanced and multi-treated disease and is a risk factor for disease progression and development of autoimmune complications. Moreover, B-lymphocytes from patients with autoimmunity produced high levels of TGFβ. These findings suggest that interactions between the B cell receptor and TLRs signaling, and an abnormal cytokine pattern play a critical role in the maintenance of self-tolerance.