The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases

ten Oever, Jaap; Kox, Matthijs; van de Veerdonk, Frank L; Mothapo, Khutso M; Slavcovici, Adriana; Jansen, Tim L; Tweehuysen, Lieke; Giamarellos-Bourboulis, Evangelos J; Schneeberger, Peter M; Wever, Peter C; Stoffels, Monique; Simon, Anna; van der Meer, Jos WM; Johnson, Melissa D; Kullberg, Bart-Jan; Pickkers, Peter; Pachot, Alexandre; Joosten, Leo AB; Netea, Mihai G

The first group consisted of patients with infectious diseases in whom plasma was obtained ≤24 hours after presentation: viral lower respiratory tract infections (LRTI) (n = 25; Table 3 ), measles [15] (n = 43), infectious mononucleosis caused by either Epstein-Barr virus (EBV) [16] or cytomegalovirus (CMV) infection (n = 16), bacterial and viral respiratory co-infections (n = 20; Table 3 ), bacterial sepsis [17] , stratified into sepsis, severe sepsis and septic shock [18] (n = 156), and candidemia [19] (n = 26). The second group comprised of patients with non-infectious inflammation: Crohn's disease (n = 15), gout (n = 36), autoinflammatory syndromes (n = 15), rheumatoid arthritis (n = 20), and pancreatitis [20] (n = 22). Patients with autoinflammatory syndromes consisted mainly of patients with well-known, genetically confirmed autoinflammatory diseases, like hyperimmunoglobulin-D syndrome, familial Mediterranean fever, Muckle-Wells syndrome and tumor necrosis factor receptor-1 associated syndrome (TRAPS). Pancreatitis was of biliary origin in 45%, none developed necrosis and all had negative blood cultures. Samples were taken ≤24 hours after presentation, except for Crohn's disease and reumatoid arthritis. Those were taken during an exacerbation of the disease ( Table 1 ). The disease activity of rheumatoid arthritis was measured with Disease Activity Score (DAS) in 28 joints (DAS28) [21] . The definition of active rheumatoid arthritis is a DAS28 > 3.2. The mean DAS28 was 4.49 (range 3.40-6.40, of whom 7 patients had a score >5.1, indicating high disease activity). Cytokine and sTLR2 and sTLR4 measurement sTLR2 and sTLR4 concentrations were measured by a commercial ELISA kits (USCN Life Science, Inc., Wuhan, China) with a lower limit of detection of 0.312 ng/ml and 0.156 ng/ml, respectively. A commercial ELISA (Sanquin, Amsterdam, The Netherlands) with a minimal detection level of 1.56 pg/ml was used for the determination of Interleukin (IL)-6 concentrations in supernatants. IL-6, tumor necrosis factor (TNF)α, IL-1Ra and IL-10 concentrations in plasma were determined using a Luminex assay (Bio-plex cytokine assay, BioRad, Hercules, CA), with a sensitivity of 6 pg/ml, 20 pg/ml, 72 pg/ml and 6 pg/ml, respectively. CRP concentrations were measured with a commercial ELISA, with a lower detection limit of 5 mg/l (IBL International, Hamburg, Germany). Samples were diluted when appropriate.