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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 direct activation of naive B-cells in vitro. (A) Enriched splenic CD19+ B-cells (2 × 106) were cultured in the presence of media, PCEP (10 μg/mL) or lipopolysaccharide (LPS) (0.1 μg/mL), and culture supernatants were collected after 48 h for quantification of IL-6 and IgM by ELISA; (B) enriched naive B-cells (2 × 105 cells/well) were co-cultured with irradiated splenocytes in the presence of medium, PCEP (5 μg/mL, 10 μg/mL and 25 μg/mL) and LPS (0.1 μg/mL) for five days. PCEP-specific B-cell proliferative responses were measured by 3H-thymidine incorporation. Results are expressed as stimulation indexes (counts per minute (cpm) in the stimulated cultures/cpm in the controls). Tests were carried out in triplicate. A stimulation index of ≥3 was considered positive for proliferative responses (above dashed lines). All of the ELISA data were statistically analyzed 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-f004: PCEP induces direct activation of naive B-cells in vitro. (A) Enriched splenic CD19+ B-cells (2 × 106) were cultured in the presence of media, PCEP (10 μg/mL) or lipopolysaccharide (LPS) (0.1 μg/mL), and culture supernatants were collected after 48 h for quantification of IL-6 and IgM by ELISA; (B) enriched naive B-cells (2 × 105 cells/well) were co-cultured with irradiated splenocytes in the presence of medium, PCEP (5 μg/mL, 10 μg/mL and 25 μg/mL) and LPS (0.1 μg/mL) for five days. PCEP-specific B-cell proliferative responses were measured by 3H-thymidine incorporation. Results are expressed as stimulation indexes (counts per minute (cpm) in the stimulated cultures/cpm in the controls). Tests were carried out in triplicate. A stimulation index of ≥3 was considered positive for proliferative responses (above dashed lines). All of the ELISA data were statistically analyzed 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: MACS isolated, enriched CD19+ B-cells were cultured in the presence of PCEP, and the culture supernatants were analyzed for cytokine and IgM responses. PCEP stimulated significant production of IgM in a dose-dependent manner, with the highest production when used at 5 µg/mL, suggesting the direct activation of enriched naive B-cells (Figure 4A). In addition, PCEP induced significant production of IL-6; however, the amounts of IL-6 produced was low (Figure 4A). Furthermore, PCEP did not induce IL-10 and IL-12 production by enriched naive B-cells (data not shown). To determine whether PCEP can induce B-cell proliferation directly, we performed in vitro B-cell proliferation assays using LPS as a positive control. PCEP did not induce positive proliferation responses in enriched B-cells (Figure 4B) at any concentrations. The data suggest that although PCEP induces direct activation of naive B-cells, but does not induce B-cell proliferative responses.


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 direct activation of naive B-cells in vitro. (A) Enriched splenic CD19+ B-cells (2 × 106) were cultured in the presence of media, PCEP (10 μg/mL) or lipopolysaccharide (LPS) (0.1 μg/mL), and culture supernatants were collected after 48 h for quantification of IL-6 and IgM by ELISA; (B) enriched naive B-cells (2 × 105 cells/well) were co-cultured with irradiated splenocytes in the presence of medium, PCEP (5 μg/mL, 10 μg/mL and 25 μg/mL) and LPS (0.1 μg/mL) for five days. PCEP-specific B-cell proliferative responses were measured by 3H-thymidine incorporation. Results are expressed as stimulation indexes (counts per minute (cpm) in the stimulated cultures/cpm in the controls). Tests were carried out in triplicate. A stimulation index of ≥3 was considered positive for proliferative responses (above dashed lines). All of the ELISA data were statistically analyzed 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-f004: PCEP induces direct activation of naive B-cells in vitro. (A) Enriched splenic CD19+ B-cells (2 × 106) were cultured in the presence of media, PCEP (10 μg/mL) or lipopolysaccharide (LPS) (0.1 μg/mL), and culture supernatants were collected after 48 h for quantification of IL-6 and IgM by ELISA; (B) enriched naive B-cells (2 × 105 cells/well) were co-cultured with irradiated splenocytes in the presence of medium, PCEP (5 μg/mL, 10 μg/mL and 25 μg/mL) and LPS (0.1 μg/mL) for five days. PCEP-specific B-cell proliferative responses were measured by 3H-thymidine incorporation. Results are expressed as stimulation indexes (counts per minute (cpm) in the stimulated cultures/cpm in the controls). Tests were carried out in triplicate. A stimulation index of ≥3 was considered positive for proliferative responses (above dashed lines). All of the ELISA data were statistically analyzed 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: MACS isolated, enriched CD19+ B-cells were cultured in the presence of PCEP, and the culture supernatants were analyzed for cytokine and IgM responses. PCEP stimulated significant production of IgM in a dose-dependent manner, with the highest production when used at 5 µg/mL, suggesting the direct activation of enriched naive B-cells (Figure 4A). In addition, PCEP induced significant production of IL-6; however, the amounts of IL-6 produced was low (Figure 4A). Furthermore, PCEP did not induce IL-10 and IL-12 production by enriched naive B-cells (data not shown). To determine whether PCEP can induce B-cell proliferation directly, we performed in vitro B-cell proliferation assays using LPS as a positive control. PCEP did not induce positive proliferation responses in enriched B-cells (Figure 4B) at any concentrations. The data suggest that although PCEP induces direct activation of naive B-cells, but does not induce B-cell proliferative responses.

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.