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Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response.

Sartorius R, D'Apice L, Trovato M, Cuccaro F, Costa V, De Leo MG, Marzullo VM, Biondo C, D'Auria S, De Matteis MA, Ciccodicola A, De Berardinis P - EMBO Mol Med (2015)

Bottom Line: A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed.We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9.Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Council of Research, Naples, Italy.

No MeSH data available.


Related in: MedlinePlus

fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/− mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/− mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-I CD8+ T cells after 72 h of co-culture with 1 × 104 DCs isolated from C57BL/6 mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fdOVA/sc-αDEC bacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-I CD8+ T cells. As a control, the proliferation of OT-I CD8+ T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6 mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).Data information: The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.
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fig03: fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/− mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/− mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-I CD8+ T cells after 72 h of co-culture with 1 × 104 DCs isolated from C57BL/6 mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fdOVA/sc-αDEC bacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-I CD8+ T cells. As a control, the proliferation of OT-I CD8+ T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6 mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).Data information: The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.

Mentions: The involvement of the TLR pathway in the activity induced by fdsc-αDEC particles after their internalization and/or degradation in the endolysosomal compartments was initially investigated by analyzing the production of the pro-inflammatory cytokine IL-6 in MyD88−/− DCs pulsed with LPS-free phage virions. As illustrated in Fig3A, the production of IL-6 by fdsc-αDEC was totally abolished in MyD88−/− DCs compared to wild-type BMDCs, confirming the involvement of the TLR pathway. Since the filamentous phage particles contain a single-strand (ss) DNA rich in unmethylated CpG sequences, and since TLR9 recognizes unmethylated CpG motifs of bacterial and viral ssDNA, we next specifically investigated the role of TLR9 in the induction of cytokine production after phage uptake.


Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response.

Sartorius R, D'Apice L, Trovato M, Cuccaro F, Costa V, De Leo MG, Marzullo VM, Biondo C, D'Auria S, De Matteis MA, Ciccodicola A, De Berardinis P - EMBO Mol Med (2015)

fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/− mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/− mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-I CD8+ T cells after 72 h of co-culture with 1 × 104 DCs isolated from C57BL/6 mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fdOVA/sc-αDEC bacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-I CD8+ T cells. As a control, the proliferation of OT-I CD8+ T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6 mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).Data information: The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.
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fig03: fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/− mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/− mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-I CD8+ T cells after 72 h of co-culture with 1 × 104 DCs isolated from C57BL/6 mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fdOVA/sc-αDEC bacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-I CD8+ T cells. As a control, the proliferation of OT-I CD8+ T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6 mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).Data information: The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.
Mentions: The involvement of the TLR pathway in the activity induced by fdsc-αDEC particles after their internalization and/or degradation in the endolysosomal compartments was initially investigated by analyzing the production of the pro-inflammatory cytokine IL-6 in MyD88−/− DCs pulsed with LPS-free phage virions. As illustrated in Fig3A, the production of IL-6 by fdsc-αDEC was totally abolished in MyD88−/− DCs compared to wild-type BMDCs, confirming the involvement of the TLR pathway. Since the filamentous phage particles contain a single-strand (ss) DNA rich in unmethylated CpG sequences, and since TLR9 recognizes unmethylated CpG motifs of bacterial and viral ssDNA, we next specifically investigated the role of TLR9 in the induction of cytokine production after phage uptake.

Bottom Line: A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed.We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9.Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Biochemistry, National Council of Research, Naples, Italy.

No MeSH data available.


Related in: MedlinePlus