Acute lung injury (ALI) caused by lipopolysaccharide (LPS) is a common, severe clinical syndrome. Injury caused by inflammation and oxidative stress in vascular endothelial and alveolar epithelial cells is a vital process in the pathogenesis of ALI. Toll-like receptor 9 (TLR9) is highly expressed in LPS-induced ALI rats. In this study, Beas-2B human pulmonary epithelial cells and A549 alveolar epithelial cells were stimulated by LPS, resulting in the upregulation of TLR9 in a concentrationdependent manner. Furthermore, TLR9 overexpression and interference vectors were transfected before LPS administration to explore the role of TLR9 in LPS-induced ALI in vitro. The findings revealed that inhibition of TLR9 reduced inflammation and oxidative stress while suppressing apoptosis of LPS-induced Beas-2B and A549 cells, whereas TLR9 overexpression aggravated these conditions. Moreover, TLR9 inhibition resulted in downregulated protein expression of myeloid differentiation protein 88 (MyD88) and activator activator protein 1 (AP-1), as well as phosphorylation of nuclear factor-?B (NF-kappaB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The phosphorylation of extracellular-regulated protein kinases 1/2 was upregulated compared to that of cells subjected to only LPS administration, and this was reversed by TLR9 overexpression. These results indicate that inhibition of TLR9 plays a protective role against LPS-induced inflammation and oxidative stress in Beas-2B and A549 cells, possibly via the MyD88/NF-kappaB and MyD88/MAPKs/AP-1 pathways.
- MeSH
- akutní poškození plic * chemicky indukované metabolismus prevence a kontrola MeSH
- epitelové buňky patologie MeSH
- krysa rodu Rattus MeSH
- lipopolysacharidy * metabolismus toxicita MeSH
- NF-kappa B metabolismus MeSH
- oxidační stres MeSH
- protein MyD88 metabolismus MeSH
- signální transdukce MeSH
- toll-like receptor 4 metabolismus MeSH
- toll-like receptor 9 genetika metabolismus MeSH
- transkripční faktor AP-1 metabolismus MeSH
- zánět chemicky indukované MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lipopolysacharidy * MeSH
- Myd88 protein, rat MeSH Prohlížeč
- NF-kappa B MeSH
- protein MyD88 MeSH
- Tlr9 protein, rat MeSH Prohlížeč
- toll-like receptor 4 MeSH
- toll-like receptor 9 MeSH
- transkripční faktor AP-1 MeSH
One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)-the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury.
- Klíčová slova
- axon regeneration, compression, early endosomes, mitochondrial DNA, sciatic nerve, the endoplasmic reticulum, transection,
- MeSH
- krysa rodu Rattus MeSH
- nemoci sedacího nervu imunologie metabolismus patologie terapie MeSH
- neurony cytologie imunologie MeSH
- potkani Wistar MeSH
- přirozená imunita imunologie MeSH
- spinální ganglia cytologie MeSH
- toll-like receptor 9 genetika metabolismus MeSH
- transkripční faktor STAT3 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Stat3 protein, rat MeSH Prohlížeč
- Tlr9 protein, rat MeSH Prohlížeč
- toll-like receptor 9 MeSH
- transkripční faktor STAT3 MeSH
Tumor Necrosis Factor Receptor 2 (TNFR2) expression is increasingly being linked to tolerogenic immune reactions and cells with suppressor function including a subset of T-regulatory cells. B-regulatory cells play an important role in control of T-cell responses and inflammation. Recently, we described TNFR2 as a marker for IL-10-producing B cells, a hallmark of this cell subset. Here, we demonstrate that proliferation of T cells is reduced in the presence of TNFR2 positive human memory B cells generated with TLR9 ligand, while TNFR2- and TNFR2+CD27- B cells display costimulatory activity. Our data further reveal that IL-10 secretion is characteristic of IgM+ naïve and memory B cells but suppressive activity is not restricted to IL-10: (i) the inhibitory effect of TNFR2+ switched memory B cells was comparable to that exerted by TNFR2+ IgM+ memory B cells although IL-10 secretion levels in the cocultures were lower; (ii) supernatants from TNFR2+ memory B cells failed to suppress T-cell proliferation. Based on our findings, we propose that formation of Breg is a specific characteristic of human memory B cells undergoing terminal differentiation. Our data further corroborate that TNFR2 represents a viable marker for identification of memory B cells with regulatory function.
- Klíčová slova
- B cells, Breg, IL-10, TLR9, TNFR2,
- MeSH
- aktivace lymfocytů genetika imunologie MeSH
- B-lymfocyty imunologie metabolismus MeSH
- běžná variabilní imunodeficience etiologie metabolismus MeSH
- buněčná diferenciace imunologie MeSH
- cytokiny metabolismus MeSH
- imunologická paměť * MeSH
- imunomodulace genetika MeSH
- interleukin-10 metabolismus MeSH
- lidé MeSH
- mezibuněčná komunikace imunologie MeSH
- receptory TNF - typ II genetika metabolismus MeSH
- regulace genové exprese * MeSH
- regulační B-lymfocyty imunologie metabolismus MeSH
- studie případů a kontrol MeSH
- T-lymfocyty imunologie metabolismus MeSH
- toll-like receptor 9 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokiny MeSH
- interleukin-10 MeSH
- receptory TNF - typ II MeSH
- TLR9 protein, human MeSH Prohlížeč
- toll-like receptor 9 MeSH
Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCβ. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.
- Klíčová slova
- Wallerian degeneration, chemokines, cytokines, damage-associated molecular patterns, disintegration, mitochondria, receptors,
- MeSH
- chlorochin farmakologie MeSH
- krysa rodu Rattus MeSH
- N-formylmethionin-leucyl-fenylalanin farmakologie MeSH
- nádorové buněčné linie MeSH
- neurilemom metabolismus patologie MeSH
- receptory CCR2 genetika metabolismus MeSH
- receptory CXCR4 genetika metabolismus MeSH
- receptory pro formylované peptidy antagonisté a inhibitory genetika metabolismus MeSH
- Schwannovy buňky cytologie účinky léků metabolismus MeSH
- signální transdukce účinky léků MeSH
- toll-like receptor 9 antagonisté a inhibitory genetika metabolismus MeSH
- upregulace účinky léků MeSH
- zánět metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorochin MeSH
- N-formylmethionin-leucyl-fenylalanin MeSH
- receptory CCR2 MeSH
- receptory CXCR4 MeSH
- receptory pro formylované peptidy MeSH
- toll-like receptor 9 MeSH
BACKGROUND: The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS: We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS: Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.
- Klíčová slova
- Cytosolic DNA sensors, DNA, Monocytes, Neutrophil extracellular traps, STING, TBK1, TLR9, Type 1 diabetes,
- MeSH
- CpG ostrůvky genetika MeSH
- cytokiny metabolismus MeSH
- diabetes mellitus 1. typu genetika metabolismus MeSH
- dítě MeSH
- DNA metabolismus MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- mladiství MeSH
- monocyty metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- signální transdukce fyziologie MeSH
- studie případů a kontrol MeSH
- toll-like receptor 9 metabolismus MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokiny MeSH
- DNA MeSH
- membránové proteiny MeSH
- protein-serin-threoninkinasy MeSH
- TBK1 protein, human MeSH Prohlížeč
- toll-like receptor 9 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.
- 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
High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.
- MeSH
- antigeny nádorové metabolismus MeSH
- antitumorózní látky aplikace a dávkování MeSH
- cytotoxicita imunologická MeSH
- dendritické buňky cytologie MeSH
- docetaxel MeSH
- experimentální nádory farmakoterapie terapie MeSH
- hydrostatický tlak MeSH
- imunitní systém MeSH
- imunoterapie metody MeSH
- infekce papilomavirem farmakoterapie terapie MeSH
- interferon gama metabolismus MeSH
- interleukin-12 metabolismus MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty farmakoterapie metabolismus terapie MeSH
- protinádorové vakcíny chemie MeSH
- slezina imunologie MeSH
- taxoidy aplikace a dávkování MeSH
- toll-like receptor 9 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antigeny nádorové MeSH
- antitumorózní látky MeSH
- docetaxel MeSH
- interferon gama MeSH
- interleukin-12 MeSH
- protinádorové vakcíny MeSH
- taxoidy MeSH
- Tlr9 protein, mouse MeSH Prohlížeč
- toll-like receptor 9 MeSH
OBJECTIVE: Dendritic cells (DCs) play an important role in pathogenesis of autoimmunity, including type 1 diabetes (T1D). In this study, we investigated DC subpopulations and their responses to TLR stimulation in T1D patients and their relatives. METHODS: We analyzed the frequency of myeloid (mDCs) and plasmacytoid DCs (pDCs) in 97 T1D patients (69 onset, 28 long-term), 67 first-degree relatives, and 64 controls. We additionally tested the IFN-alpha production by pDCs upon stimulation with TLR 7, 8 and 9 agonists. RESULTS: A lower number of mDCs and pDCs were found in T1D patients and their relatives. Of all the tested TLR ligands, only stimulation with CpG 2216 induced IFN-alpha production that was the highest in T1D relatives, except of autoantibody-negative relatives bearing the protective haplotypes. CONCLUSION: Our data demonstrate disturbances in DC number and function expressed most significantly in T1D relatives and point to a potential role of TLR9-induced IFN-alpha production in T1D development.
- Klíčová slova
- Dendritic cell;, Interferon-alpha;, Type 1 diabetes,
- MeSH
- dendritické buňky imunologie metabolismus MeSH
- diabetes mellitus 1. typu imunologie metabolismus MeSH
- dítě MeSH
- dospělí MeSH
- interferon alfa biosyntéza krev MeSH
- leukocyty mononukleární účinky léků metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- mladiství MeSH
- mladý dospělý MeSH
- oligodeoxyribonukleotidy farmakologie MeSH
- počet buněk MeSH
- předškolní dítě MeSH
- rodina MeSH
- toll-like receptor 9 metabolismus MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CpG ODN 2216 MeSH Prohlížeč
- interferon alfa MeSH
- ligandy MeSH
- oligodeoxyribonukleotidy MeSH
- toll-like receptor 9 MeSH
