Dysfunction of Toll-Like Receptor-2, −4, and −6: Is There a Role in the Pathogenesis of Immunodeficiency in Multiple Myeloma?.
Authors: Terpos Evangelos , Dimopoulos Meletios A. , Xirakia Charoula , Christoulas Dimitrios , Anagnostopoulos Athanasios , Katodritou Eirini , Zervas Kostas , Sideras Paschalis , Tsionos Konstantinos , Andreakos Evangelos 
Published: October 2019

Abstract
Infections are a major cause of death in multiple myeloma (MM). There is limited information for the possible role of innate immunity in the pathogenesis of immunodeficiency in MM. Innate immune detection of pathogens relies on specific classes of microbial sensors, such as Toll-like receptors (TLRs) that detect structural patterns, which do not exist in the host. The aim of this study was to evaluate the expression and function of TLRs in newly diagnosed patients with MM. According to our knowledge, such information is not available in the literature. Thirty-two MM patients at diagnosis (18M/14F; median age 69 years), 6 MGUS patients and 14 healthy, age- and gender-matched controls were studied. Seven patients had stage 1, 14 stage 2 and 11 stage 3 myeloma, according to ISS. After the collection of peripheral blood, mononuclear cells (PBMCs) were isolated by Ficoll centrifugation (Histopaque-1077; Sigma-Aldrich). These cells were measured for the expression of TLRs (antibodies from eBioscience) using fluorescence activated flow cytometry (FC 500, Beckman Coulter). In addition, 1x106 cells/ml were cultured in 5% FCS 1% pen/strep RPMI in the presence or absence of various TLR ligands and supernatants collected after 20h. These were examined for the presence of inflammatory cytokines (tumor necrosis-alpha, TNF-α; and interleukin-6, IL-6) by ELISA (Becton Dickinson). We found that although patients with MM express TLRs in PBMCs, their response to certain TLR ligands is defective when compared to healthy controls. TLR2, TLR4 and TLR6 of PBMCs of healthy controls reacted to the presence of their respective ligands PAM3CYS (gram positive and negative bacterial), LPS (gram negative) and FLT-1 (gram positive) producing TNF-α at a median value of 2.4 ng/mL (range: 1–3 ng/ml), while their action in patients with MM was significantly reduced (median value of TNF-α: 190 pg/ml; range 100–500 pg/ml; p<0.001). The reduced response of TLR4 in MM patients was independent of the LPS concentration used. On the contrary, TLR7 and TLR8 from MM reacted normally to their ligand R-848 (Imiquimob) to secrete high levels of TNF-α (median value for patients and controls: 4.5 and 4.2 ng/ml, respectively; p=NS). NOD1, another pattern recognition receptor that recognizes bacterial peptidoglycans also reacted normally in MM. Similar observations have been made for IL-6 expression. There was no difference in terms of TLRs function between MGUS patients and controls and between myeloma patients of different disease stages. In seven MM patients, cells were pre-treated with bortezomib for 30 min before addition of the TLR ligand. Bortezomib administration abrogated TLR 7/8 response in PBMCs even at very low concentrations (1nM). This study suggests that there is a significant defect in TLR function in MM, especially of these involved in immunity against bacterial infections. Thus the immune system fails to receive early priming signal, which may contribute to the increased rate of infections observed in MM. The restoration of function of TLRs to their normal levels has the potential to improve bacterial immunity in MM.