Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

• Klíčová slova
• B cell-like receptor signaling, MEK1/2, blood dendritic cell antigen 2, c-FOS, plasmacytoid dendritic cells, regulatory receptors, toll-like receptors 7 and 9 (TLR7/9), type I interferon,
• MeSH
• B-lymfocyty imunologie   MeSH
• buněčné linie MeSH
• dendritické buňky fyziologie   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• interferon typ I metabolismus   MeSH
• lidé MeSH
• MAP kinasa-kinasa 1 metabolismus   MeSH
• MAP kinasa-kinasa 2 metabolismus   MeSH
• MAP kinasový signální systém MeSH
• NF-kappa B metabolismus   MeSH
• proteinkinasa C metabolismus   MeSH
• protoonkogenní proteiny c-fos metabolismus   MeSH
• receptory antigenů B-buněk genetika   metabolismus   MeSH
• toll-like receptor 9 metabolismus   MeSH
• vápníková signalizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• extracelulárním signálem regulované MAP kinasy MeSH
• interferon typ I MeSH
• MAP kinasa-kinasa 1 MeSH
• MAP kinasa-kinasa 2 MeSH
• NF-kappa B MeSH
• proteinkinasa C MeSH
• protoonkogenní proteiny c-fos MeSH
• receptory antigenů B-buněk MeSH
• toll-like receptor 9 MeSH

The innate immune cells sense microbial infection and self-ligands by pathogen recognition receptors (PRRs), such as toll-like receptors (TLRs) and regulatory receptors (RRs), associated with immunoreceptor tyrosine-based activation motif (ITAM). Rapid activation and concerted action of PRRs signaling and feedback inhibitory mechanisms must be engaged to ensure the host defense functions and to prevent cytotoxicity associated with excessive activation. ITAM-associated RRs can generate stimulatory or, paradoxically, inhibitory signals. The network of ITAM-associated RR, together with TLR-signaling pathways, are responsible for immunogenic or tolerogenic responses of macrophages and dendritic cells to their microenvironment. In macrophages, TLR4 signaling is inhibited by low-avidity ligation of ITAM-associated receptors, while high-avidity ligation of ITAM-associated receptors results in potentiation of TLR4 signaling together with resistance to extracellular cytokine microenvironment signals. In contrast to macrophages, TLR7/9 signaling in plasmacytoid DCs (pDCs) is inhibited by high-avidity ligation of ITAM-associated RR, while low-avidity ligation does not show any effect. Surprisingly, interference of ITAM-associated receptor signaling with TLR pathways has not been reported in conventional dendritic cells. Here, we present an overview of molecular mechanisms acting at the crossroads of TLR and ITAM-signaling pathways and address the question of how the high-avidity engagement of the ITAM-associated receptors in pDCs inhibits TLR7/9 signaling. Cellular context and spatiotemporal engagement of ITAM- and TLR-signaling pathways are responsible for different outcomes of macrophage versus pDC activation. While the cross-regulation of cytokine and TLR signaling, together with antigen presentation, are the principal functions of ITAM-associated RR in macrophages, the major role of these receptors in pDCs seems to be related to inhibition of cytokine production and reestablishment of a tolerogenic state following pDC activation. Pharmacologic targeting of TLR and ITAM signaling could be an attractive new therapeutic approach for treatment of chronic infections, cancer, and autoimmune and inflammatory diseases related to pDCs.

• Klíčová slova
• B cell receptor-like signaling, conventional dendritic cells, immunoreceptor tyrosine-based activation motif-associated receptor, macrophage, plasmacytoid dendritic cell, regulatory receptors, toll-like receptors,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/β and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function-IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.

• MeSH
• cytokiny metabolismus   MeSH
• dendritické buňky účinky léků   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kinasa Syk antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• přirozená imunita MeSH
• signální transdukce účinky léků   fyziologie   MeSH
• toll-like receptory agonisté   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• inhibitory proteinkinas MeSH
• kinasa Syk MeSH
• toll-like receptory MeSH

To target the E7 protein of human papilloma virus 16 to the cell surface, a fusion gene was constructed. It encodes the signal peptide, part of the immunoglobulin (IgG)-like domain, the transmembrane anchor of vaccinia virus (VV) hemagglutinin (HA), and the complete E7-coding sequence. The fusion gene was expressed under the HA late promoter by a recombinant VV, designated VV-E7-HA. The E7-HA protein was displayed on the surface of cells infected with the recombinant virus and was more stable than unmodified E7. The biological properties of the VV-E7-HA virus were compared with those of a VV-E7 virus that expressed the unmodified E7 and with a VV expressing the Sig-E7-LAMP fusion protein. While the first two of these recombinants were based on VV strain Praha, the third was derived from the WR strain of VV. Infection of mice with the VV-E7-HA virus induced the formation of E7-specific antibodies with the predominance of the IgG2a isotype, whereas the other two viruses did not induce the formation of E7-specific antibodies. Unlike the other two viruses, VV-E7-HA did not induce a response of cytotoxic T lymphocytes or Th1 cells and did not protect mice against the growth of E7-expressing tumors. Thus, VV-E7-HA induced a differently polarized immune response to the E7 protein than the other two viruses.

• MeSH
• buněčná imunita MeSH
• experimentální nádory prevence a kontrola   MeSH
• hemaglutininy virové imunologie   MeSH
• interleukin-4 biosyntéza   MeSH
• králíci MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• onkogenní proteiny virové analýza   imunologie   MeSH
• Papillomavirus E7 - proteiny MeSH
• protilátky virové biosyntéza   MeSH
• rekombinantní fúzní proteiny imunologie   MeSH
• vakcinace MeSH
• virové vakcíny imunologie   MeSH
• virus vakcinie genetika   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemaglutininy virové MeSH
• interleukin-4 MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• onkogenní proteiny virové MeSH
• Papillomavirus E7 - proteiny MeSH
• protilátky virové MeSH
• rekombinantní fúzní proteiny MeSH
• virové vakcíny MeSH