Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy Noémie Caillot, PhD, Julien Bouley, PhD, Karine.

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Presentation transcript:

Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy Noémie Caillot, PhD, Julien Bouley, PhD, Karine Jain, MSc, Sandrine Mariano, PhD, Sonia Luce, MSc, Stéphane Horiot, MSc, Sabi Airouche, MSc, Chloé Beuraud, PhD, Christian Beauvallet, PhD, Philippe Devillier, MD, Sylvie Chollet-Martin, PhD, Christine Kellenberger, PhD, Laurent Mascarell, PhD, Henri Chabre, PhD, Thierry Batard, PhD, Emmanuel Nony, PhD, Vincent Lombardi, PhD, Véronique Baron-Bodo, PhD, Philippe Moingeon, PhD Journal of Allergy and Clinical Immunology Volume 140, Issue 3, Pages 759-770.e13 (September 2017) DOI: 10.1016/j.jaci.2016.10.036 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Levels of acidic forms of FetA in pretreatment sera from patients with grass pollen allergy correlate with sublingual immunotherapy clinical efficacy. A, Two-dimensional gel image of serum proteins, with localization of FetA proteoforms. MW, Molecular weight. B, Volumes of FetA proteoforms in spots 439 and 469 from sera of ARs relative to that of ANRs. C and D, Spot volumes are shown as boxplots in the active (Fig 1, C) and placebo (Fig 1, D) groups (left panels). *P < .05, **P < .01, or ***P < .001. Correlations of acidic protein spot volumes with ARTSS improvement in patients from the active (Fig 1, C) and placebo (Fig 1, D) groups (right panels) are shown. E, Abundances of acidic FetA spots were assessed by using 2D-DiGE before and after sublingual immunotherapy in sera from patients receiving placebo or active tablets. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Sialylation of FetA on O-linked glycans. A, Deconvoluted MS spectrum of FetA (A chain) purified from human serum. Mass differences corresponding to monosaccharides or disaccharides are indicated on the spectrum. B, Two-dimensional gel image of a mixture of sialylated FetA (labeled in green with Cy2) and AsialoFetA (labeled in red with Cy3). MW, Molecular weight. C, Composition of OGs determined by using LC-MS/MS. PTM, Posttranslational modification. Yellow squares, N-acetylglucosamine; yellow circles, hexose; purple diamonds, Neu5Ac (sialic acid). Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Levels of O-linked sialylated FetA in pretreatment sera from patients with grass pollen allergy. A and B, OG abundances are expressed in arbitrary units (AU) and as boxplots in the active (Fig 3, A) and placebo (Fig 3, B) groups. *P < .05 or ***P < .001. Correlations of OG3 abundance with ARTSS improvement in patients from the active (Fig 3, A) and placebo (Fig 3, B) groups (right panels) after sublingual immunotherapy are shown. C, Receiver operating characteristic (ROC) analysis of OG3 abundance in serum samples from patients of the active group. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 In vivo silencing of the gene encoding FetA enhances OVA-induced allergic inflammation in BALB/c mice. A, FetA silencing in OVA-sensitized animals was achieved by means of injections of FetA-specific vivo-morpholino. B, FetA concentrations in sera are expressed as mean values relative to D-2 values normalized to 100 (n = 3 mice per group). *P < .05. C, Airway hyperresponsiveness measured by using whole-body plethysmography is expressed as an enhanced pause (Penh) index (means ± SEMs, n = 6 mice per group). D, Lung resistance (Rl) was assessed by using invasive plethysmography (means ± SEMs, n = 6 mice per group). *P < .05 (*) or ****P < .0001. E, Cytokine secretion was assessed in splenocytes from animals untreated or treated with morpholinos (means ± SEMs, n = 6 mice per group). **P < .01. F, Identification by using LC-MS/MS of sialic acid–containing OGs associated with murine FetA. Yellow squares, N-acetylglucosamine; yellow circles, hexose; open diamonds, N-glycolylneuraminic acid (Neu5Gc). Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 FetA, but not AsialoFetA, synergizes with LPS in a TLR4-dependent pathway to enhance the proallergic profile of human MoDCs. A and B, TLR4 activation was measured in human TLR4 HEK-293 reporting cells. #P < .05. C, Expression of costimulatory molecules. D and E, Cytokine production (Fig 5, D) and gene expression (Fig 5, E) by MoDCs cultured in the presence of either FetA or AsialoFetA. Data are shown as means ± SEMs (n = 6 to 8 donors tested; Fig 5, C-E). F and G, Cytokine production was measured in immature MoDCs cultured in the presence of either FetA or AsialoFetA plus either a 5-grass-pollen (Fig 5, F) or D farinae allergen extract (Fig 5, G). Results are presented as means ± SEMs (n = 6 donors tested). *P < .05, ** P < .01, or ****P < .0001. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 FetA and AsialoFetA bind LPS but do not influence its physical binding to TLR4–MD-2. A and B, Surface plasmon resonance analyses of the interaction between injected LPS and FetA (Fig 6, A) or AsialoFetA (Fig 6, B) immobilized on CM5 sensor chips. C-E, Sensorgrams obtained after injection of either LPS (Fig 6, C), a combination of LPS with FetA (Fig 6, D) or LPS with AsialoFetA (Fig 6, E) over TLR4–MD-2 immobilized on a CM5 sensor chip. Sensorgrams are represented as response units (RUs) at specified times in seconds. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 VO56.07A study design. Schematic design of the pollen chamber trial. After screening and randomization, 82 patients with grass pollen allergy were treated sublingually once daily with either a placebo (n = 40) or a grass pollen (n = 42) tablet for 4 months. Grass pollen challenges were performed before treatment (at inclusion), at day 7, and at months 1, 2, and 4 during AIT, with an evaluation of ARTSS during the 4 hours after challenge. The search for predictive biomarkers was performed on serum samples collected from patients before treatment initiation. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Immunoaffinity enrichment of human FetA. Visualization of human FetA by using SDS-PAGE after SYPRO protein staining (A) and Western blot analyses (B). Lanes 1 and 2, 1.5 μg of crude RPMI medium supplemented with 10% human serum before and after affinity chromatography, respectively; lane 3, 0.3 μg of commercial FetA (BioVendor, Brno, Czech Republic); lane 4, 0.6 μg of FetA obtained by immunoaffinity purification; lane M, molecular weight markers. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Human FetA (B chain) contains sialylated O-linked carbohydrates. A, LC-UV analysis of the reduced forms of natural FetA (see deconvoluted mass spectrum of the A chain in Fig 2, A). B-D, MS analysis of FetA B chain. Nonglycosylated (Fig E3, B) and sialylated forms (on O-linked glycans) are observed in the mass spectra (Fig E3, C and D). Yellow squares, N-acetylglucosamine; yellow circles, hexose; purple diamonds, Neu5Ac (sialic acid). Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Confirmation of human FetA desialylation by using MS. A, LC-UV analysis of the reduced forms (by using 20 mmol/L DTT) of natural AsialoFetA obtained after treatment of purified native (ie, sialylated) FetA with neuraminidase. B-D, MS analyses of A (Fig E4, B) and B chains (Fig E4, C and D) from FetA and/or AsialoFetA. Yellow squares, N-acetylglucosamine; yellow circles, hexose. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Correlation between OG3 peptide abundance and spot volumes (spots 428 + 439 + 448) in pretreatment sera of individual patients. Spearman correlation results obtained at the individual patient level on pretreatment blood samples from both placebo and active cohorts, relating FetA OG3 abundance measured by using MS and FetA spot volumes assessed by using 2D-DiGE, are shown. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Timothy grass pollen sensitization model with concomitant FetA gene silencing. A, Naive mice were sensitized intraperitoneally with a timothy grass pollen extract adsorbed on alum. At days 7 and 8, mice received an intranasal administration of a timothy grass pollen extract. Mice received intravenous injections of either specific vivo-morpholinos or controls at days −1, 2, 5, and 8 to silence FetA expression. i.n., Intranasal; i.p., intraperitoneal; i.v., intravenous. B, Airway hyperresponsiveness determined by using whole-body plethysmography was measured in response to increasing concentrations of methacholine, with results expressed as an enhanced pause (Penh) index as means ± SEMs (n = 6 mice per group). * P < .05, ** P < .01, and **** P < .0001. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Comparison of amino acid sequences of human and murine FetA. Pairwise alignment of amino acid sequences of human (UniProtKB entry P02765) and murine (UniprotKB entry P29699) FetA performed with UniProt alignment tool. O-glycosylation sites were confirmed by using MS. Journal of Allergy and Clinical Immunology 2017 140, 759-770.e13DOI: (10.1016/j.jaci.2016.10.036) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions