107, 102C108 [PubMed] [Google Scholar] 9. after 12 h was also seen when cells were stimulated for control Cdh15 with LPS but not with BSA (Fig. 2< 0.001; **, <0.01; *, <0.05. Strikingly, the treatment with parthenolide abolished the induction of cytokine genes by exosomes as decided for IL-6, TNF-, and IL-1 by RT-PCR (Fig. 3emphasized that an LPS contamination was unlikely to be the source of triggering. Our data rather suggested that a protein determinant associated with exosomes is responsible for the induction of p65 phosphorylation. Open in a separate window Physique 4. Proteinase-sensitive determinant(s) on exosomes trigger THP-1 cells. added to Lysyl-tryptophyl-alpha-lysine THP-1 cells to exclude unspecific effects. TLRs Are Involved in Exosome-mediated Signaling It is known that TLRs can activate the NFB pathway. Indeed, we noticed that the effects of exosomes on cell signaling were similar to TLR agonists. This prompted us to study more closely the role of TLRs in exosome-mediated signaling. THP-1 cells expressed TLR2 and TLR4 but were unfavorable for TLR7 and TLR8 as detected by RT-PCR (Fig. 5= 3 experiments are shown. ***, < 0.001; **, <0.01; *, <0.05. Lysyl-tryptophyl-alpha-lysine = 4 are shown. STAT3 phosphorylation was examined by Western blotting. in the absence of any selective pressure from the immune system. Contrary to exosomes from cell culture, we assumed that exosomes from body fluid are more likely to reflect the situation. Thus, in the current study, a main goal was to deepen the understanding of immune cell stimulation by (25). We also provide evidence that activation of the NFB and STAT3 pathways were necessary for the induction of cytokine genes. Collectively, these data provide novel insights into the signaling potential of exosomes. We also show that in THP-1 cells the TLRs are key Lysyl-tryptophyl-alpha-lysine receptors for exosome-mediated signaling. This is based on the following findings: (i) the stable knockdown of TLR2 or TLR4 led to a partial reduction of cytokine gene induction and release; (ii) antibodies to TLR2 and TLR4 alone could block in part the phosphorylation of STAT3 and subsequent Lysyl-tryptophyl-alpha-lysine induction of IL-1 and IL-6 transcription, but the effect was strongest when both antibodies were used in combination; (iii) human exosomes could trigger secretion of cytokines in mouse DCs and macrophages, but this was abolished in cells deficient for MyD88, an adaptor protein required for TLR signaling. Our results confirm and extend previous work demonstrating a functional role of TLR2 (25, 30). For the first time we also show an involvement of TLR4. Previous studies have focused mostly on mouse systems and tissue culture-derived exosomes, and a role of TLR4 was not investigated. Meanwhile Fabbri reported that miRNAs in exosomes can trigger the endosomal TLR7/8 leading to cytokine secretion (31). Due to the absence of these receptors in THP-1 cells we were unable to investigate this. Our data do not exclude the possibility that in addition to TLRs other molecules can serve as exosomal receptors on monocytic or other immune cells. An important question is usually which determinants on exosomes trigger TLRs and cytokine production. Previous studies have reported conflicting results. Xiang proposed that exosomes isolated from grown breast adenocarcinomas were able to induce expansion of MDSCs via a mechanism dependent on the exosomal presence of prostaglandin E2 (43). Chalmin used cell-culture derived exosomes and found that activation of MDSCs was dependent on the presence of HSP72 on exosomes whereas no exosomal prostaglandin E2 was found in their study (25). Using body fluid-derived exosomes we observed that the stimulating potential was destroyed by proteinase K but not with DNase or RNase treatment, supporting the notion that signals come from proteins. These determinant(s) need to be further characterized. It should be borne in mind that beside HSPs other alarmins including HMGB1 or.