Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Published byVerawati Lesmono Modified over 5 years ago

Similar presentations

Presentation on theme: "A fusion protein of flagellin and ovalbumin suppresses the TH2 response and prevents murine intestinal allergy  Stefan Schülke, PhD, Manja Burggraf, MSc,"— Presentation transcript:

1 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 e12 (December 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

16 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

17 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

18 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  , e12DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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

Similar presentations

Toll-like receptor 2 ligands promote chronic atopic dermatitis through IL-4–mediated suppression of IL-10  Susanne Kaesler, PhD, Thomas Volz, MD, Yuliya.

Toll-like receptor 2 ligands promote chronic atopic dermatitis through IL-4–mediated suppression of IL-10  Susanne Kaesler, PhD, Thomas Volz, MD, Yuliya.
Cor a 1–reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen–associated food allergy to hazelnut  Claudia.

Cor a 1–reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen–associated food allergy to hazelnut  Claudia.
Barbara Stanic, PhD, Willem van de Veen, PhD, Oliver F

Barbara Stanic, PhD, Willem van de Veen, PhD, Oliver F
Cheng-Ming Sun, Edith Deriaud, Claude Leclerc, Richard Lo-Man  Immunity 

Cheng-Ming Sun, Edith Deriaud, Claude Leclerc, Richard Lo-Man  Immunity 
Therapeutic reversal of food allergen sensitivity by mature retinoic acid–differentiated dendritic cell induction of LAG3+CD49b−Foxp3− regulatory T cells 

Therapeutic reversal of food allergen sensitivity by mature retinoic acid–differentiated dendritic cell induction of LAG3+CD49b−Foxp3− regulatory T cells 
IgE-mediated systemic anaphylaxis and impaired tolerance to food antigens in mice with enhanced IL-4 receptor signaling  Clinton B. Mathias, PhD, Suejy.

IgE-mediated systemic anaphylaxis and impaired tolerance to food antigens in mice with enhanced IL-4 receptor signaling  Clinton B. Mathias, PhD, Suejy.
Induction of anergic allergen-specific suppressor T cells using tolerogenic dendritic cells derived from children with allergies to house dust mites 

Induction of anergic allergen-specific suppressor T cells using tolerogenic dendritic cells derived from children with allergies to house dust mites 
Let-7 microRNA–mediated regulation of IL-13 and allergic airway inflammation  Manish Kumar, MSc, Tanveer Ahmad, MSc, Amit Sharma, PhD, Ulaganathan Mabalirajan,

Let-7 microRNA–mediated regulation of IL-13 and allergic airway inflammation  Manish Kumar, MSc, Tanveer Ahmad, MSc, Amit Sharma, PhD, Ulaganathan Mabalirajan,
Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening  Theresa Thalhamer, PhD, Heidi Dobias, MSc, Tatjana.

Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening  Theresa Thalhamer, PhD, Heidi Dobias, MSc, Tatjana.
IL-25 and CD4+ TH2 cells enhance type 2 innate lymphoid cell–derived IL-13 production, which promotes IgE-mediated experimental food allergy  Jee-Boong.

IL-25 and CD4+ TH2 cells enhance type 2 innate lymphoid cell–derived IL-13 production, which promotes IgE-mediated experimental food allergy  Jee-Boong.
Maternal allergy increases susceptibility to offspring allergy in association with TH2- biased epigenetic alterations in a mouse model of peanut allergy 

Maternal allergy increases susceptibility to offspring allergy in association with TH2- biased epigenetic alterations in a mouse model of peanut allergy 
Allergy prevention starts before conception: Maternofetal transfer of tolerance protects against the development of asthma  Tobias Polte, PhD, Christian.

Allergy prevention starts before conception: Maternofetal transfer of tolerance protects against the development of asthma  Tobias Polte, PhD, Christian.
Cor a 1–reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen–associated food allergy to hazelnut  Claudia.

Cor a 1–reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen–associated food allergy to hazelnut  Claudia.
Toll-like receptor 7–induced naive human B-cell differentiation and immunoglobulin production  Mark C. Glaum, MD, PhD, Shilpi Narula, MD, Decheng Song,

Toll-like receptor 7–induced naive human B-cell differentiation and immunoglobulin production  Mark C. Glaum, MD, PhD, Shilpi Narula, MD, Decheng Song,
Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy  Raphaela.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy  Raphaela.
Selective control of SIRP-α–positive airway dendritic cell trafficking through CD47 is critical for the development of TH2-mediated allergic inflammation 

Selective control of SIRP-α–positive airway dendritic cell trafficking through CD47 is critical for the development of TH2-mediated allergic inflammation 
Ting-ting Zhang, MSc, Klaus Okkenhaug, PhD, Baher F

Ting-ting Zhang, MSc, Klaus Okkenhaug, PhD, Baher F
Frank Kirstein, PhD, Natalie E

Frank Kirstein, PhD, Natalie E
Protective role of nuclear factor of activated T cells 2 in CD8+ long-lived memory T cells in an allergy model  Roman Karwot, PhD, Joachim H. Maxeiner,

Protective role of nuclear factor of activated T cells 2 in CD8+ long-lived memory T cells in an allergy model  Roman Karwot, PhD, Joachim H. Maxeiner,
Elisabeth Roesler, MSc, Richard Weiss, PhD, Esther E

Elisabeth Roesler, MSc, Richard Weiss, PhD, Esther E

Similar presentations

Ads by Google