Challenges with various TLR ligands (TLRLs)in combination with D-galactosamine (GalN) in rodents may mimic diverse conditions of acute inflammation and organ failure. Here, we report that CpG (ODN1826, TLR9 agonist)/GalN induced a liver-specific injury with modest systemic effects, whereas R848 (resiquimod, TLR7/8 agonist)/GalN exhibited systemic and liver toxicity. We also observed the protective effect of Gr-1+ cells (the population containing neutrophils) against liver injury in both the R848/GalN and CpG/GalN models. In cytokine measurements, the intraperitoneal administration of antibodies showed a non-specific tolerance induction effect, which was more pronounced in the CpG/GalN than in the R848/GalN model. Cytokine analyses also suggested that the TLR9 agonist/GalN induced a limited degree of systemic inflammation compared to TLR7/8 agonist/GalN models. The relevance of this finding to the TLR9-mediated induction of stress tolerance (protective effect) in non-immune cells is discussed.

• MeSH
• galaktosamin toxicita   MeSH
• imidazoly toxicita   MeSH
• lékové postižení jater etiologie   metabolismus   patologie   MeSH
• lipopolysacharidy toxicita   MeSH
• membránové glykoproteiny agonisté   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• stupeň závažnosti nemoci MeSH
• toll-like receptor 7 agonisté   MeSH
• toll-like receptor 8 agonisté   MeSH
• toll-like receptor 9 agonisté   MeSH
• zánět chemicky indukované   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

• MeSH
• buňky Hep G2 MeSH
• chronická hepatitida B virologie   MeSH
• cytokiny metabolismus   MeSH
• hepatocyty virologie   MeSH
• interferon alfa metabolismus   MeSH
• kruhová DNA metabolismus   MeSH
• kultivační média speciální farmakologie   MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• přirozená imunita účinky léků   MeSH
• replikace viru účinky léků   MeSH
• systémy cílené aplikace léků MeSH
• toll-like receptory agonisté   metabolismus   MeSH
• virus hepatitidy B fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Publikační typ
• časopisecké články MeSH

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

• MeSH
• basigin genetika   imunologie   MeSH
• biologické markery metabolismus   MeSH
• delfíni rodu Stenella genetika   imunologie   virologie   MeSH
• exprese genu MeSH
• genetická predispozice k nemoci * MeSH
• histokompatibilita - antigeny třídy II genetika   imunologie   MeSH
• infekce viry z rodu Morbillivirus genetika   imunologie   virologie   MeSH
• jednonukleotidový polymorfismus * MeSH
• molekuly buněčné adheze genetika   imunologie   MeSH
• Morbillivirus imunologie   patogenita   MeSH
• Phocoena genetika   imunologie   virologie   MeSH
• proteiny přenášející kationty genetika   imunologie   MeSH
• receptor 1 spouštějící přirozenou cytotoxicitu genetika   imunologie   MeSH
• SLAMF1 protein genetika   imunologie   MeSH
• toll-like receptor 3 genetika   imunologie   MeSH
• toll-like receptor 7 genetika   imunologie   MeSH
• toll-like receptor 8 genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články 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

Due to the persisting threat of development of new highly pathogenic influenza A subtypes, a mucosal vaccination which would induce a potent and cross-protective reaction is desirable. We succeeded in mucosal immunization of mice with an inactivated influenza A virus by using delipidated Bacillus firmus (DBF) as adjuvant. The mechanism of adjuvant effect was followed in NALT by comparing the response after intranasal immunization by inactivated influenza virus type A (H1N1) alone, adjuvant alone (DBF), or by a mixture of virus+DBF. Expression of selected gene groups was tested via qPCR at 7 different time-points: cytokines (IL-2, IFN-γ, IL-4, IL-6, and IL-10), type I interferons (IFN-α4, IFN-α11, IFN-α12, and IFN-β), toll-like receptors (TLR2, TLR3, TLR7, and TLR9), iNOS and CCR7. Intranasally administered DBF and the mixture of virus+DBF induced an elevated expression of IFN-γ, IL-6 and IL-10 cytokines, type I interferons, iNOS, and pDC markers in NALT. Multimarker qPCR data was analyzed by relative quantification and by principal component analysis. DBF has been shown to be a very efficient adjuvant for the stimulation of innate immunity after IN immunization. DBF accelerated, increased, and prolonged the antiviral response.

• MeSH
• adjuvancia imunologická aplikace a dávkování   MeSH
• analýza hlavních komponent MeSH
• aplikace intranazální MeSH
• Bacillus imunologie   MeSH
• časové faktory MeSH
• cytokiny genetika   MeSH
• exprese genu účinky léků   MeSH
• imunizace metody   MeSH
• interferon typ I genetika   MeSH
• interleukin-10 genetika   MeSH
• interleukin-2 genetika   MeSH
• lymfoidní tkáň metabolismus   MeSH
• membránové glykoproteiny genetika   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nazofarynx metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• synergismus léků MeSH
• toll-like receptor 2 genetika   MeSH
• toll-like receptor 3 genetika   MeSH
• toll-like receptor 7 genetika   MeSH
• toll-like receptor 9 genetika   MeSH
• toll-like receptory genetika   MeSH
• vakcíny proti chřipce aplikace a dávkování   imunologie   MeSH
• virus chřipky A, podtyp H1N1 imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH