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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.


Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) induces robust secretion of IL-1β and IL-18 in splenic dendritic cells (DCs). Enriched splenic DCs from BALB/c mice were incubated for 12 h with media, PCEP (50 μg/mL), alum (40 mg/mL), lipopolysaccharide (LPS) (0.1 μg/mL), PCEP+LPS or alum+LPS. Supernatants were collected for measuring IL-1β and IL-18 by ELISA, and the cell extracts were analyzed for pro-IL-1β by western blotting. (A) Secretion of IL-1β and IL-18 in enriched splenic DCs; (B) pro-caspase-1 (45 kDa), pro-IL-1β (31 kDa) and β-actin (43 kDa) detection in cell lysates by western blot analysis. Data was analyzed by one-way ANOVA, and the comparisons between the treatments were done by Tukey’s multiple comparison test; *** p < 0.0001.
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vaccines-02-00500-f001: Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) induces robust secretion of IL-1β and IL-18 in splenic dendritic cells (DCs). Enriched splenic DCs from BALB/c mice were incubated for 12 h with media, PCEP (50 μg/mL), alum (40 mg/mL), lipopolysaccharide (LPS) (0.1 μg/mL), PCEP+LPS or alum+LPS. Supernatants were collected for measuring IL-1β and IL-18 by ELISA, and the cell extracts were analyzed for pro-IL-1β by western blotting. (A) Secretion of IL-1β and IL-18 in enriched splenic DCs; (B) pro-caspase-1 (45 kDa), pro-IL-1β (31 kDa) and β-actin (43 kDa) detection in cell lysates by western blot analysis. Data was analyzed by one-way ANOVA, and the comparisons between the treatments were done by Tukey’s multiple comparison test; *** p < 0.0001.

Mentions: Stimulation of enriched splenic DCs with PCEP induced significantly higher IL-1β and IL-18 secretion relative to media control and alum. In addition, stimulation with PCEP in the presence of LPS triggered significantly higher secretion of IL-1β and IL-18 compared to PCEP or LPS alone (Figure 1A). Further, in the presence of LPS, PCEP induced significantly higher secretion of IL-1β and IL-18 compared to the alum+LPS combination (Figure 1A).


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)

Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) induces robust secretion of IL-1β and IL-18 in splenic dendritic cells (DCs). Enriched splenic DCs from BALB/c mice were incubated for 12 h with media, PCEP (50 μg/mL), alum (40 mg/mL), lipopolysaccharide (LPS) (0.1 μg/mL), PCEP+LPS or alum+LPS. Supernatants were collected for measuring IL-1β and IL-18 by ELISA, and the cell extracts were analyzed for pro-IL-1β by western blotting. (A) Secretion of IL-1β and IL-18 in enriched splenic DCs; (B) pro-caspase-1 (45 kDa), pro-IL-1β (31 kDa) and β-actin (43 kDa) detection in cell lysates by western blot analysis. Data was analyzed by one-way ANOVA, and the comparisons between the treatments were done by Tukey’s multiple comparison test; *** p < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

vaccines-02-00500-f001: Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) induces robust secretion of IL-1β and IL-18 in splenic dendritic cells (DCs). Enriched splenic DCs from BALB/c mice were incubated for 12 h with media, PCEP (50 μg/mL), alum (40 mg/mL), lipopolysaccharide (LPS) (0.1 μg/mL), PCEP+LPS or alum+LPS. Supernatants were collected for measuring IL-1β and IL-18 by ELISA, and the cell extracts were analyzed for pro-IL-1β by western blotting. (A) Secretion of IL-1β and IL-18 in enriched splenic DCs; (B) pro-caspase-1 (45 kDa), pro-IL-1β (31 kDa) and β-actin (43 kDa) detection in cell lysates by western blot analysis. Data was analyzed by one-way ANOVA, and the comparisons between the treatments were done by Tukey’s multiple comparison test; *** p < 0.0001.
Mentions: Stimulation of enriched splenic DCs with PCEP induced significantly higher IL-1β and IL-18 secretion relative to media control and alum. In addition, stimulation with PCEP in the presence of LPS triggered significantly higher secretion of IL-1β and IL-18 compared to PCEP or LPS alone (Figure 1A). Further, in the presence of LPS, PCEP induced significantly higher secretion of IL-1β and IL-18 compared to the alum+LPS combination (Figure 1A).

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.