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Caspase-1 Dependent IL-1β Secretion and Antigen-Specific T-Cell Activation by the Novel Adjuvant, PCEP.

Awate S, Eng NF, Gerdts V, Babiuk LA, Mutwiri G - Vaccines (Basel) (2014)

Bottom Line: Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1β and IL-18 secretion in response to PCEP stimulation.Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation.We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

View Article: PubMed Central - PubMed

Affiliation: Vaccinology and Immunotherapeutics program, School of Public Health, 107 Wiggins Road, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. sunita.awate@usask.ca.

ABSTRACT
The potent adjuvant activity of the novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), with various antigens has been reported previously. However, very little is known about its mechanisms of action. We have recently reported that intramuscular injection of PCEP induces NLRP3, an inflammasome receptor gene, and inflammatory cytokines, including IL-1β and IL-18, in mouse muscle tissue. Caspase-1 is required for the processing of pro-forms of IL-1β and IL-18 into mature forms and is a critical constituent of the NLRP3 inflammasome. Hence, in the present study, we investigated the role of caspase-1 in the secretion of IL-1β and IL-18 in PCEP-stimulated splenic dendritic cells (DCs). Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1β and IL-18 secretion in response to PCEP stimulation. Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation. However, PCEP directly activated B-cells to induce significant production of IgM. In addition, PCEP+ovalbumin (OVA) immunized mice showed significantly increased production of antigen-specific IFN-γ by CD4⁺ and CD8⁺ T-cells. We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

No MeSH data available.


PCEP induces antigen-specific IFN-γ production in CD4+ and CD8+ T-cells. BALB/c mice were immunized i.m. with 25 μL each of either phosphate-buffered saline (PBS) as a control, 10 µg ovalbumin (OVA) or 50 μg of PCEP co-delivered with 10 μg OVA. Booster immunization was given on Day 14 to half of the mice in each group. Mice were euthanized on Days 9 and 21 after the first immunization to collect spleens. Splenocytes (1 × 106 cells) were restimulated with OVA (10 μg/mL) in culture for 12 h. Intracellular production of IFN-γ by CD8+ (A) and CD4+ (B) T-cells was analyzed by flow cytometry. Statistical analysis was done by one-way ANOVA, and the differences between the treatments were compared by Tukey’s multiple-comparison test; ***p < 0.0001, **p < 0.001, *p < 0.05.
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vaccines-02-00500-f005: PCEP induces antigen-specific IFN-γ production in CD4+ and CD8+ T-cells. BALB/c mice were immunized i.m. with 25 μL each of either phosphate-buffered saline (PBS) as a control, 10 µg ovalbumin (OVA) or 50 μg of PCEP co-delivered with 10 μg OVA. Booster immunization was given on Day 14 to half of the mice in each group. Mice were euthanized on Days 9 and 21 after the first immunization to collect spleens. Splenocytes (1 × 106 cells) were restimulated with OVA (10 μg/mL) in culture for 12 h. Intracellular production of IFN-γ by CD8+ (A) and CD4+ (B) T-cells was analyzed by flow cytometry. Statistical analysis was done by one-way ANOVA, and the differences between the treatments were compared by Tukey’s multiple-comparison test; ***p < 0.0001, **p < 0.001, *p < 0.05.

Mentions: To evaluate antigen-specific T-cell responses induced by PCEP, BALB/c mice were immunized with PCEP co-delivered with OVA. As shown in Figure 5, intracellular IFN-γ production was increased in mice immunized with PCEP+OVA compared with OVA alone. The frequencies of IFN-γ+ CD8+ T-cells on Day 9 (3.6% vs. 1.3%) and Day 21 (6.5% vs. 4.2%) were significantly higher in mice immunized with PCEP + OVA than in mice immunized with OVA alone (Figure 5A). Similarly, the frequencies of IFN-γ+ CD4+ T-cells on Day 9 (9.0% vs. 3.8%) and Day 21 (7.3% vs. 5.3%) were significantly higher in mice immunized with PCEP + OVA than in mice immunized with OVA alone (Figure 5B). These results indicate that PCEP induces antigen-specific activation of CD8+ and CD4+ T-cells.


Caspase-1 Dependent IL-1β Secretion and Antigen-Specific T-Cell Activation by the Novel Adjuvant, PCEP.

Awate S, Eng NF, Gerdts V, Babiuk LA, Mutwiri G - Vaccines (Basel) (2014)

PCEP induces antigen-specific IFN-γ production in CD4+ and CD8+ T-cells. BALB/c mice were immunized i.m. with 25 μL each of either phosphate-buffered saline (PBS) as a control, 10 µg ovalbumin (OVA) or 50 μg of PCEP co-delivered with 10 μg OVA. Booster immunization was given on Day 14 to half of the mice in each group. Mice were euthanized on Days 9 and 21 after the first immunization to collect spleens. Splenocytes (1 × 106 cells) were restimulated with OVA (10 μg/mL) in culture for 12 h. Intracellular production of IFN-γ by CD8+ (A) and CD4+ (B) T-cells was analyzed by flow cytometry. Statistical analysis was done by one-way ANOVA, and the differences between the treatments were compared by Tukey’s multiple-comparison test; ***p < 0.0001, **p < 0.001, *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494223&req=5

vaccines-02-00500-f005: PCEP induces antigen-specific IFN-γ production in CD4+ and CD8+ T-cells. BALB/c mice were immunized i.m. with 25 μL each of either phosphate-buffered saline (PBS) as a control, 10 µg ovalbumin (OVA) or 50 μg of PCEP co-delivered with 10 μg OVA. Booster immunization was given on Day 14 to half of the mice in each group. Mice were euthanized on Days 9 and 21 after the first immunization to collect spleens. Splenocytes (1 × 106 cells) were restimulated with OVA (10 μg/mL) in culture for 12 h. Intracellular production of IFN-γ by CD8+ (A) and CD4+ (B) T-cells was analyzed by flow cytometry. Statistical analysis was done by one-way ANOVA, and the differences between the treatments were compared by Tukey’s multiple-comparison test; ***p < 0.0001, **p < 0.001, *p < 0.05.
Mentions: To evaluate antigen-specific T-cell responses induced by PCEP, BALB/c mice were immunized with PCEP co-delivered with OVA. As shown in Figure 5, intracellular IFN-γ production was increased in mice immunized with PCEP+OVA compared with OVA alone. The frequencies of IFN-γ+ CD8+ T-cells on Day 9 (3.6% vs. 1.3%) and Day 21 (6.5% vs. 4.2%) were significantly higher in mice immunized with PCEP + OVA than in mice immunized with OVA alone (Figure 5A). Similarly, the frequencies of IFN-γ+ CD4+ T-cells on Day 9 (9.0% vs. 3.8%) and Day 21 (7.3% vs. 5.3%) were significantly higher in mice immunized with PCEP + OVA than in mice immunized with OVA alone (Figure 5B). These results indicate that PCEP induces antigen-specific activation of CD8+ and CD4+ T-cells.

Bottom Line: Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1β and IL-18 secretion in response to PCEP stimulation.Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation.We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

View Article: PubMed Central - PubMed

Affiliation: Vaccinology and Immunotherapeutics program, School of Public Health, 107 Wiggins Road, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada. sunita.awate@usask.ca.

ABSTRACT
The potent adjuvant activity of the novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), with various antigens has been reported previously. However, very little is known about its mechanisms of action. We have recently reported that intramuscular injection of PCEP induces NLRP3, an inflammasome receptor gene, and inflammatory cytokines, including IL-1β and IL-18, in mouse muscle tissue. Caspase-1 is required for the processing of pro-forms of IL-1β and IL-18 into mature forms and is a critical constituent of the NLRP3 inflammasome. Hence, in the present study, we investigated the role of caspase-1 in the secretion of IL-1β and IL-18 in PCEP-stimulated splenic dendritic cells (DCs). Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1β and IL-18 secretion in response to PCEP stimulation. Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation. However, PCEP directly activated B-cells to induce significant production of IgM. In addition, PCEP+ovalbumin (OVA) immunized mice showed significantly increased production of antigen-specific IFN-γ by CD4⁺ and CD8⁺ T-cells. We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

No MeSH data available.