In order to understand the mechanisms leading to impaired functionality

of chronically activated DCs we determined the kinetics and extent of the LPS induced IL-12, TNF and IL-6 gene expression in MoDCs developed from peripheral blood monocytes in a 2-day culture in the presence or absence of 5 ng/mL LPS. We used this relatively low LPS concentration as it did not induce a strong DC activation measured at the level of inflammatory cytokines or the expression of CD86 and CD83 at day 2 but it consistently induced a desensitization of developing MoDCs to further LPS-mediated activation (Fig. 1A). Thus inhibitory signals contributing to DC inactivation may not be obscured by a strong DC activation. We analyzed MoDC activation following click here a short, 2-day culture, to better represent https://www.selleckchem.com/products/BAY-73-4506.html an in vivo situation when monocyte precursors enter inflamed tissues and differentiate to DCs in the presence of activation

signals that readily induce effector functions. At day 2 we observed the induction of CD1a and CD209 (DC-SIGN) and the downregulation of CD14 on a high proportion of developing MoDCs underlying the hypothesis that monocytes are able to obtain DC phenotype in such short period (Supporting Information Fig. 1). As Fig. 1B shows, a 2-day LPS pre-treatment completely blocked the induction of IL-12, TNF and IL-6 genes by a second LPS stimulus whereas, without LPS pre-treatment MoDCs responded to LPS signal with 3-mercaptopyruvate sulfurtransferase a rapid and strong induction of these genes. To study if the tolerization of developing MoDCs by an early encounter with stimulatory signals is a general phenomenon, or if it is specific for single LPS stimulus, we treated the cells with a wide variety of stimulatory factors, applied separately or in combination with LPS between day 0 and 2 of MoDC cultures. Few of these signals induced detectable TNF production when applied to

monocytes alone, namely, heat-killed Staphylococcus aureus (HKSA), an inducer of TLR2 signals and CL075 that triggers TLR7/8 (Fig. 1C). LPS synergistically increased the levels of TNF when combined with CD40L, the TLR2 ligands HKSA or Pam3Cys, with CL075 or with the combination of TNF, IL-1 and IL-6. No activation or very low cytokine levels were observed with TNF, IFN-γ and the TLR3 ligand poly(I:C). Despite the strong initial MoDC activation induced by several types of stimuli, when the cells were washed and reactivated by 100 ng/mL LPS at day 2, we observed a complete inhibition of TNF production in MoDCs that differentiated in the presence of CD40L, HKSA, Pam3Cys, CL075, TNF or the combination of TNF, IL-1 and IL-6 (Fig. 1C, right panel). The 48 h presence of LPS resulted in a persistent DC inactivation both when LPS was added alone and when it was combined with any of the other activation signals.