A novel lectin, isolated from basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1-10 [micro]g/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48h resulted in extensive up-regulation of co-stimulatory molecules CD80, CD86, as well as the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a Th1-polarized response in naive CD4+CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as IL-6, IL-8 and TNF-[alpha]. By using a specific TLR4 signaling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 Nf-kB and p38 MAPK phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in Nf-kB activity, with less effect on the induction of p38 MAPK signaling than that of LPS-matured DCs. CNL had the ability to activate human DCs in such a way as to subsequently direct Th1-type T cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies, with aims such as to strengthen anti-tumor immune responses.
COBISS.SI-ID: 3099761
Monocyte-derived dendritic cells (DCs) are considered an indispensible and one of primary tools for in vitro DC-based studies. For majority of in vitro DC-based studies the medium of choice is supplemented RPMI, with certain variable ingredients such as HEPES buffer or Phenol Red (PHR). In effort to identify potential obstruction of DC differentiation process due to presence of mentioned additives, we differentiated DCs using RPMI either with or without HEPES or PHR. Although PHR caused a certain down-regulation of immature DCs (iDCs) differentiation markers and lower expression of co-stimulatory molecules on mature DCs, these changes were not significant. In contrast, use of RPMI also containing HEPES resulted in significantly lower CD1a and DC-SIGN expression on iDCs and extensively lowered co-stimulatory molecule expression after DC activation (HEPES-DCs). Furthermore, DCs differentiated in HEPES-free RPMI possessed more genuine immature/mature DC characteristics in context of Th1 polarization. Additionally, during classical differentiation procedure, fewer DCs remained adherent and possessed better overall morphology in HEPES-free medium. In summary our study clarifies a seemingly minor, but a very important issue, that will most likely facilitate lab work for many scientists dealing with monocyte-derived DCs.
COBISS.SI-ID: 999999
Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists,inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.
COBISS.SI-ID: 2971761