A fusion protein of flagellin and ovalbumin suppresses the TH2 response and prevents murine intestinal allergy Stefan Schülke, PhD, Manja Burggraf, MSc,

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Presentation transcript:

A fusion protein of flagellin and ovalbumin suppresses the TH2 response and prevents murine intestinal allergy Stefan Schülke, PhD, Manja Burggraf, MSc, Zoe Waibler, PhD, Andrea Wangorsch, BSc, Sonja Wolfheimer, Ulrich Kalinke, PhD, Stefan Vieths, PhD, Masako Toda, PhD, Stephan Scheurer, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 6, Pages 1340-1348.e12 (December 2011) DOI: 10.1016/j.jaci.2011.07.036 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression of TLR5 on murine mDCs. BALB/c CD11b+CD11c+B220− mDCs were stimulated for 24 hours with LPS (10 μg/mL), rOVA (10 μg/mL), rflaA (6.9 μg/mL), rflaA (6.9 μg/mL) plus rOVA (10 μg/mL), or rflaA:OVA (16.9 μg/mL; A) or with increasing doses of rflaA (B) and analyzed for TLR5 expression by means of FACS. Gray-shaded areas, Unstimulated; black lines, stimulated as indicated; black dotted lines, isotype control. Results show representative data from one of 2 independent experiments. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 rflaA:OVA fusion protein induces IL-2 production and represses IL-4 and IFN-γ production from OVA-specific T cells. BALB/c mDCs were cultured with (black bars) or without (gray bars) DO11.10 CD4+ T cells and stimulated with rOVA, rflaA, rflaA plus rOVA, or rflaA:OVA. IL-2, IL-6, IL-4, IL-10, and IFN-γ content in the culture supernatants were quantified by means of ELISA. Results are representative data ± SDs taken from one of 3 independent experiments. n.d., Not detectable. ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 rflaA:OVA suppresses TH1 and TH2 cytokine secretion by OVA-specific TH2-biased T cells. CD4+ T cells from OVA-immunized BALB/c mice were cocultured with BALB/c mDCs and stimulated with rOVA, rflaA:OVA, or both for 72 hours. Levels of the TH2 cytokines IL-4 (A) and IL-5 (B) and the TH1 cytokine IFN-γ (C) in culture supernatants were quantified by means of ELISA. Results are means ± SDs of 2 independent experiments. ∗P > .05, ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 rflaA:OVA-induced cytokine secretion by mDCs depends on TLR signaling. Comparison of cytokine secretion by mDCs derived from C57BL/6 (black bars) or MyD88−/− (gray bars) mice cultured without (A) or with (B) OT-II CD4+ T cells. Cell cultures were stimulated with LPS (10 μg/mL), rOVA (10 μg/mL), rflaA (6.9 μg/mL), rflaA (6.9 μg/mL) plus rOVA (10 μg/mL), or rflaA:OVA (16.9 μg/mL) for 72 hours. Levels of IL-6, IL-10, and IFN-γ in the culture supernatants were quantified by means of ELISA. Results are means ± SDs of 2 independent experiments. n.d., Not detectable. ∗∗P > .01, ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 rflaA:OVA suppresses IFN-γ secretion by naive OVA-specific T cells. C57BL/6 mDCs were cocultured with OT-II CD4+ T cells and stimulated with equimolar amounts of rOVA (white bars), rflaA plus rOVA (gray bars), rflaA:OVA (black bars), or the indicated amount of LPS for 72 hours, and culture supernatants were analyzed for levels of IFN-γ. Results are means ± SDs of 3 independent experiments. ∗P > .05. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Prophylactic vaccination with rflaA:OVA prevents intestinal allergy. Control (PBS → PBS → CN) and OVA-sensitized (OVA/A) mice were continuously challenged with either OVA-containing pellets (EW) or normal pellets (CN) for 6.5 days (A). Mean symptom scores (B) and body weights normalized to the individual body weight before challenge and mean core body temperatures (C) were analyzed (n = 8 mice per group). i.p., Intraperitoneal. ∗P > .05, ∗∗P > .01. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Prophylactic vaccination with rflaA:OVA increases IgG2a/IgE ratios. Sera of control (PBS → PBS → CN) and OVA-sensitized mice (OVA/A) were collected at the end of OVA pellet challenge (day 6.5) and analyzed for OVA-specific IgE (A), IgG2a (B), and IgG1 (C) antibody levels by means of ELISA. IgG2a/IgE ratios (D) were calculated for each mouse and depicted as median values per group (n = 8 mice). ∗∗P > .01. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 FACS analysis of in vitro–differentiated mDCs. Unstained samples were used to determine positive staining thresholds, and putative mDCs were gated for mDC-specific size and granularity. Subsequently, B220−CD11c+ mDCs were gated and checked for coexpression of CD11b. Expression of TLR5 on the cell surfaces of CD11b+CD11c+B220− mDCs was analyzed. FSC, Forward scatter; PE, phycoerythrin; SSC, side scatter. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 rflaA:OVA-induced IL-4 and IFN-γ production is diminished by mDC-derived IL-10. BALB/c mDCs were cultured with (black bars) or without (gray bars) DO11.10 CD4+ T cells and stimulated with rflaA:OVA for 72 hours in the presence of rIL-10 or anti–IL-10 antibodies, respectively, and analyzed for IFN-γ (A), IL-4 (B), and IL-10 (C) in the cell-culture supernatant. Results are representative data from 3 independent experiments ± SDs. ∗P > .05, ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 rflaA:OVA potently induces mDC-derived cytokine production. C57BL/6 mDCs were stimulated with increasing equimolar amounts of rOVA (white bars), rflaA plus rOVA (grey bars), rflaA:OVA (black bars), or the indicated amount of LPS (hatched bars) for 72 hours, and cell-culture supernatants were analyzed for levels of IL-10 and IL-6. Results are means of 3 independent experiments ± SDs. ∗P > .05, ∗∗P > .01, ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Symptom score used for clinical evaluation. For the determination of symptom scores, individual mice were assessed for softness of feces (scores: 0, normal; 1, soft; 2, fluid; and 3, mucus like) and phenotype (scores: 0, normal fur; 1, slightly ruffed fur; and 2, ruffed fur) on a daily basis. Partial scores were summed to obtain the overall symptom scores. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Prophylactic vaccination with rflaA:OVA prevents intestinal allergy. Control (PBS → PBS → CN) and OVA-sensitized mice (OVA/A) were continuously challenged with either OVA-containing pellets (EW) or normal pellets (CN) for 6.5 days. Mean EW pellet uptake per mouse and day was analyzed (n = 8 mice per group). Depicted are means ± SDs of each group per day. ∗P > .05. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 rflaA:OVA vaccination reduces expression of both TH2 cytokines locally in intestinal homogenates and systemic T-cell activation in the spleen. The TH2 cytokines IL-4 and IL-13 in intestinal homogenates were determined by using multiplex ELISAs (n = 8 mice per group, A). T-cell activation was investigated by measuring CD62 ligand (CD62-L) expression on splenic CD4+ and CD8+ T-cell subsets (day 5.5, B). Gray shading, Pooled T cells from untreated animals (n = 4 per group). Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Prophylactic vaccination with rflaA:OVA increases IgG2a/IgE ratios 1 week after the first sensitization. Sera of control (PBS → PBS → CN) and OVA-sensitized mice (OVA/A) were collected 1 week after the first sensitization with OVA and analyzed for OVA-specific IgE (A), IgG2a (B), and IgG1 (C) levels by means of ELISA. IgG2a/IgE ratios (D) were calculated for each mouse and depicted as median values per group (n = 8 mice). ∗∗P > .01. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 Prophylactic vaccination with rflaA:OVA increases IgG2a/IgE ratios 1 week after the second sensitization. Sera of control (PBS → PBS → CN) and OVA-sensitized (OVA/A) mice were collected on day 21 after the first sensitization with OVA and analyzed for OVA-specific IgE (A), IgG2a (B), and IgG1 (C) levels by means of ELISA. IgG2a/IgE ratios (D) were calculated for each mouse and depicted as median values per group (n = 8 mice). ∗∗∗P > .001. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E9 Vaccination with rflaA:OVA reduces mediator release from rat basophil leukemia (RBL 2H3) cells passively sensitized with sera obtained from vaccinated mice. For determination of mediator release, RBL 2H3 cells were passively sensitized with sera from vaccinated and control mice and subsequently stimulated with OVA. β-Hexosaminidase release into the supernatant was quantified in regard to the total release induced by Triton X-100. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E10 Therapeutic vaccination with rflaA:OVA reduces both symptom scores and T-cell activation. Control (PBS → PBS → CN) and OVA-sensitized mice (OVA/A) were continuously challenged with either OVA-containing pellets (EW) or normal pellets (CN) for 5.5 days (A). Mean symptom scores were analyzed (n = 6 mice per group; B). T-cell activation was analyzed by measuring CD62 ligand (CD62-L) expression on splenic CD4+ and CD8+ T-cell subsets (day 5.5). Gray shading, Pooled T cells from untreated animals (n = 3 per group; C). ∗P > .05. Journal of Allergy and Clinical Immunology 2011 128, 1340-1348.e12DOI: (10.1016/j.jaci.2011.07.036) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions