Fig 1.
IL-1β targeted vaccine (1βEPP) elicits relatively high antibody responses, decreases body weight gain, and protects from hyperglycemia in KK-Ay mice.
(A) IL-1β epitope peptide sequence. (B) Work flow and time line of 1βEPP vaccination. (C) ELISA was performed by using IL-1β epitope peptide to detect antibody titers in serum collected from the immunized mice on days 10, 24 and 38 after primary immunization. (D) ELISA was performed by using hIL-1β, mIL-1β or mIL-1α to detect antibody titers. Body weight (E) and body weight gain (F) were measured at the indicated time points. (G) Blood glucose levels were measured at different time points after overnight fasting. Data are shown as mean ± SEM (n = 8). Compared with the KK-Ay control mice, *p < 0.05, **p < 0.01.
Fig 2.
IL-1β targeted vaccine (1βEPP) improves glucose tolerance in KK-Ay mice.
(A) 1βEPP -treated KK-Ay mice were challenged with 2 g/kg body weight of glucose, and then blood glucose levels were measured at 0, 30, 60, 90, 120 and 150 min. (B) Area under the curve (AUC) for blood glucose (0–150 min) during the ipGTT was calculated. (C) Serum insulin was measured during the ipGTT at 0 and 30 min after glucose injection. (D) Insulin stimulatory index as a ratio of stimulated (30 min) over basal (0 min) serum insulin concentration. (E) HOMA-IR as a product of fasting insulin and blood glucose levels. Data are shown as mean ± SEM (n = 8). Compared with the KK-Ay control mice, *p < 0.05, **p < 0.01.
Fig 3.
IL-1β targeted vaccine (1βEPP) improves insulin secretion and insulin sensitivity in KK-Ay mice.
(A) KK-Ay mice were fasted for 5 h before ipITT was performed. Blood glucose levels were measured at 0, 30, 60, 90 and 120 min after i.p. injection of insulin (0.75 U/kg body weight). (B) AUC for blood glucose (0–120 min) was calculated during the ipITT. Data are shown as mean ± SEM (n = 8). Compared with the KK-Ay control mice, *p < 0.05.
Fig 4.
IL-1β targeted vaccine (1βEPP) restores β-cell mass and protects from β-cell apoptosis.
(A) Triple staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (red), insulin (green), and DAPI (blue) was performed on fixed, paraffin-embedded pancreas sections. (B) β-cell mass per pancreas was estimated as the relative cross-sectional area of β-cells (determined by quantifying the cross-sectional area occupied by β-cells divided by the cross-sectional area of total tissue) multiplied by the weight of the pancreas. (C) β-cell apoptosis was quantified by percentage of TUNEL-positive β-cells. Ten sections from each pancreas spanning the width of the pancreas were included in the analysis. Data are shown as mean ± SEM. Compared with the KK-Ay control mice, *p < 0.05, **p < 0.01.
Fig 5.
IL-1β targeted vaccine (1βEPP) reduces IL-1β gene expression and inhibits NF-κB activation in KK-Ay mice.
(A) mRNA levels of IL-1β in pancreas islets were measured by quantitative RT-PCR after thrice of 1βEPP vaccination. (B) Phosphorylated RelA (pRelA) (α-tubulin as a loading control) and total IKKβ (GAPDH as a loading control) levels in pancreas tissues of KK-Ay mice were detected by Western blot. Intensities of pRelA (C) and IKKβ (D) bands were quantified using ImageJ software and then normalized to α-tubulin or GAPDH, respectively. Data are shown as mean ± SEM (n = 8). Compared with the KK-Ay control mice, *p < 0.05.
Fig 6.
IL-1β targeted vaccine (1βEPP) reduces serum lipid levels in KK-Ay mice.
Serum levels of free fatty acids (A), triglycerides (B), and total cholesterol (C) were measured on day 56 after the primary immunization. Non-HDL cholesterol (D) was obtained by subtracting HDL-cholesterol from the total cholesterol. Data are shown as mean ± SEM (n = 8). Compared with the KK-Ay control mice, *p < 0.05.
Fig 7.
Bioactivity and safety assessment of 1βEPP.
(A) In vivo inflammatory activity. Groups of C57BL/6J mice were received i.p. injection of 1μg hIL-1β, IL-1β epitope peptide or s.c. injection of 50 μg 1βEPP. Three hours later, serum IL-6 levels were quantified using an IL-6 quantitative ELISA kit. (B) Effects of endogenous IL-1β on antibody titer. KK-Ay mice were immunized once with 1βEPP and subsequently i.p. injected with 1 ng lipopolysaccharide and 20 mg N-galactosamine or s.c. injected with 50 μg 1βEPP. Antibody titer was measured before injection and 10 days thereafter. (C) In vivo neutralization activity to IL-1β. After immunized with either 1βEPP or PLA for three times, KK-Ay mice were received i.p. injection of 1 μg hIL-1β, three hours later, serum IL-6 levels were quantified using an IL-6 quantitative ELISA kit. Data are shown as mean ± SEM (n = 6). *p < 0.05, ***p < 0.001.