Article Type: Original Article
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Received date: 12/17/2013
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Revised date: 06/04/2014
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Accepted date: 06/12/2014
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Title:
Accepted Article
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Functional relevance of NLRP3 inflammasome-mediated IL-1 during acute allergic airway inflammation1
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Short title:
Anakinra ameliorates allergic airway inflammation
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Authors:
Manuel Ritter1,2, Kathrin Straubinger2, Stephanie Schmidt2, Dirk H. Busch2,3,
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Stefanie Hagner4, Holger Garn4, Clarissa Prazeres da Costa2* and Laura E. Layland1,2*
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1
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Affiliations:
Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),
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University Clinic Bonn, Bonn, Germany; 2Institute of Medical Microbiology, Immunology and
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Hygiene (MIH), Technische Universität München, Munich, Germany; 3Clinical Cooperation
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Groups ‘‘Antigen-specific Immunotherapy’’ and “Immune Monitoring”, Helmholtz Center
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Munich (Neuherberg) and Technische Universität München, Munich, Germany; 4Institute of
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Laboratory Medicine and Pathobiochemistry, Medical Faculty, Philipps-University Marburg,
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Marburg, Germany.
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*equal contribution
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Corresponding Authors
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Dr. L. E. Layland
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Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Clinic
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Bonn, Sigmund Freud Strasse 25, 53105, Bonn, Germany.
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Tel: +49 228 287 11387
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[email protected];
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Dr. C. Prazeres da Costa
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Institute of Medical Microbiology, Immunology and Hygiene (MIH), Technische Universität
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München, Trogerstrasse 30, 81675, Munich, Germany; This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cei.12400
1 This article is protected by copyright. All rights reserved.
Tel: +49 89 4140 4130
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[email protected]
Accepted Article
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Keywords
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Allergy, Anakinra, IL-1, IL-1 receptor antagonist, NLRP3-inflammasome.
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Abbreviations: AAI: allergic airway inflammation; AHR: Airway hyper-responsiveness; ASC:
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apoptosis-associated speck-like protein containing a caspase recruitment domain; BAL:
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bronchoalveloar lavage; GINA: Global Initiative of Asthma; IL-1R: IL-1 receptor; IL-1Ra: IL-1
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receptor antagonist; NLRP3: nucleotide oligomerization domain (NOD)-like receptor family,
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pyrin domain 3; OVA: ovalbumin; RT: room temperature.
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2 This article is protected by copyright. All rights reserved.
Accepted Article
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Summary
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Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However,
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certain symptomatic individuals remain at risk for serious morbidity and mortality which
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prompts the identification of novel therapeutic targets and treatment strategies. Thus, using
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an adjuvant-free TH2 murine model we have deciphered the role of IL-1 signalling during
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allergic airway inflammation (AAI). Since functional IL-1 depends on inflammasome
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activation we first studied asthmatic manifestations in specific inflammasome-deficient
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(NLRP3-/- and ASC-/-) and IL-1 receptor type 1-/- (IL-1R1-/-) mice on the BALB/c background. To
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verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung
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pathology, airway hyper-responsiveness and OVA-specific immune responses. In the absence
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of NLRP3 inflammasome-mediated IL-1 release all symptoms of AAI were reduced except
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OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling
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reduced asthmatic development we administered the IL-1R antagonist Anakinra (Kineret®)
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during critical immunological time-points: sensitization or challenge. Amelioration of
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asthmatic symptoms was only observed when Anakinra was administered during OVA
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challenge. Our findings indicate that blocking IL-1 signalling could be a potential
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complementary therapy for allergic airway inflammation.
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3 This article is protected by copyright. All rights reserved.
Accepted Article
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Introduction
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Over the last decade, the importance of inflammasome mediated release of IL-1 has been
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shown in a variety of models and moreover, has provided the basis for the treatment of
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several auto-inflammatory diseases such as rheumatoid arthritis, gout and type 2 diabetes
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[1-3]. In addition, several studies have demonstrated that IL-1 plays an important role
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during bronchial asthma development. Indeed, asthmatic patients were shown to harbour
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elevated levels of IL-1 in their bronchoalveloar lavage (BAL) [4]. Other studies have further
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reported that this proinflammatory cytokine promotes the infiltration of eosinophils and
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inflammation within the lung and encourages both mast and T cell activation [5,6]. Currently,
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300 million people are estimated to be affected by asthma worldwide (GINA 2010) and the
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prevalence continues to increase. Although current therapeutic strategies, such as
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corticosteroids and long or short acting beta agonists, can control attacks and disease
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progression, a quarter of a million people still succumbed to the syndrome in 2009 (GINA
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2009) [7,8]. Asthma is characterized by dominant TH2 immune responses including enhanced
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IL-4, IL-5 and IL-13 responses, allergen-specific immunoglobulin production, eosinophilia,
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airway inflammation, bronchoconstriction and airway hyperresponsiveness (AHR) [9-11].
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Thus, when assessing the onset of asthma in murine models is it imperative to address both
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cellular and pathological aspects.
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The secretion of functional bioactive IL-1 however, depends on the inflammasome which
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mediates, via caspase-1 activation, the cleavage of the inactive cytokine precursor (pro-IL-
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1 into the active form (IL-1. In general, this multiprotein complex contains proteins such
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as central oligomerization domain (NACHT), a leucine rich repeat (LRR) domain, apoptosis4 This article is protected by copyright. All rights reserved.
associated speck-like protein containing caspase recruitment domain (ASC) and caspase-1
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[12-14]. For instance, the well-studied NLRP3 inflammasome plays an essential role in a
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variety of scenarios and is triggered by numerous pathogens such as parasites [15], fungi
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[16], bacteria [17] or danger signals such as ATP [18] or crystalline silica and asbestos
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[19,20]. However, its role during allergic asthma remains somewhat controversial. For
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example, IL-1 receptor type 1 (IL-1R1)-deficient mice present reduced allergic responses and
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lung inflammation [21,22] and ovalbumin (OVA)-treated NLRP3-/- mice exhibit severely
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dampened lung inflammation, OVA-specific immunoglobulins, cytokine responses and
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eosinophil infiltration [23]. In contrast to the latter, Allen et al. demonstrated that allergic
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airway inflammation within NLRP3-deficient mice is similar to wildtype control mice [24].
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Both of these NLRP3 inflammasome studies were performed using C57BL/6 mice which are
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renowned for their dominant TH1 based immune responses and reduced allergic airway
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inflammation when compared to TH2-biased BALB/c mice [25-28]. Therefore, we have
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assessed the role of the NLRP3 inflammasome and functional IL-1 during OVA-induced AAI
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in a BALB/c murine model and observed altered antigen-specific TH responses, reduced
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eosinophil infiltration and airway inflammation in mice deficient for NLRP3, ASC and the IL-
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1R1. To analyze the dynamics of functional IL-1 in more detail, we blocked IL-1 signalling
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using the IL-1R antagonist (IL-1Ra) Anakinra. The application of this therapy ameliorated
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allergic airway inflammation only during the challenge phase and implies that blocking IL-1
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signalling could complement current asthma treatment regimen.
Accepted Article
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5 This article is protected by copyright. All rights reserved.
Accepted Article
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Materials and Methods
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Mice
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BALB/c and C57BL/6 wildtype mice were purchased from Harlan® (Borchen, Germany). ASC-,
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NLRP3- and IL-1R1-deficient mice on BALB/c background (backcrossed at least nine
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generations) were a kind gift from Prof. Jürg Tschopp (University of Lausanne, Switzerland).
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Mice were bred under specific pathogen-free (spf) conditions in the animal facilities at the
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Institute of Medical Microbiology, Immunology and Hygiene (Munich, Germany) in
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accordance with national and EU guidelines 86/809. Experimental mice were sex- and age-
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matched and the study was approved by the Regierung von Oberbayern, Munich, Germany
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(Animal License number Az. 55.2.1.54-2532-67-12).
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OVA-induced allergic airway inflammation model and Anakinra treatment
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Female BALB/c wildtype, ASC-, NLRP3- and IL-1R1-deficient mice were thrice sensitized
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subcutaneously (s.c.) in the neck with 10µg ovalbumin (OVA) grade VI (Sigma-Aldrich,
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Taufkirchen, Germany) or PBS (control mice) on days 0, 7 and 14 without adjuvant and
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consecutively challenged by aerosol inhalation on days 26-28 with 10µg OVA grade V (Sigma-
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Aldrich). On day 31 (3 days after the last OVA challenge) mice were sacrificed and assessed
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for allergic airway development (Supporting information, Fig. S1a). For IL-1 type cytokine
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blocking experiments mice were treated with consecutive doses of Anakinra (Kineret®;
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Biovitrum, Stockholm, Sweden) during OVA-sensitization or challenge. For details see
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schemes in Supporting information, Fig. S1b,c. Experiments were performed using either
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15mg/kg (i.p), 100mg/kg (i.p.) or 150mg/kg (s.c.) in the neck per day.
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6 This article is protected by copyright. All rights reserved.
Accepted Article
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Measurement of airway hyperresponsiveness (AHR)
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Airway hyperresponsiveness (AHR) to methacholine (Sigma-Aldrich) was determined using
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the Flexivent system (SCIREQ, Montreal, Canada). Following anaesthesia the trachea was
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intubated with a 1.2 mm tracheal cannula and the lungs mechanically ventilated at a
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respiratory frequency of 150 breaths per min, a tidal volume of 10ml/kg and a positive end-
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expiratory pressure of 3ml H2O. After exposing mice to aerosolized PBS to retrieve the
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baseline value, bronchoconstriction was induced using increasing concentrations (1, 2.5, 5,
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10, 25 and 50 mg/ml in PBS) of aerosolized methacholine using an ultrasonic nebulizer.
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Dynamic resistance was recorded over 1 minute intervals (every 5 secs) after exposure to
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defined doses of methacholine via a standardized inhalation manoeuvre (SnapShot-150)
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[29,30].
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Analysis of bronchoalveolar lavage
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Mice were euthanized with Narcoren® (Merial, Halbergmoos, Germany) and lungs were
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flushed twice with 1ml PBS containing proteinase inhibitor cocktail tablets (Roche
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Diagnostics, Mannheim, Germany). The obtained BAL was weighed and centrifuged at 230g
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for 5min (4°C). Resulting supernatants were frozen at -20°C and the cytokine content
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determined using commercially available ELISA kits (eBiosciences, Frankfurt, Germany). The
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cell pellets were resuspended in PBS containing 2% FCS and counted. To analyze the
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composition of immune cells within the BAL, 150µl of cell suspension was centrifuged on
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glass slides at 400rpm for 5min using the Shandon Cytospin 3 centrifuge (Thermo Scientific®,
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Hamburg, Germany). Glass slides were dried overnight at room temperature (RT) and
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stained using the Diff-Quick staining set (Medion Diagnostics, Langen, Germany) according to 7 This article is protected by copyright. All rights reserved.
manufacturer’s instructions. Cell differentiation was performed as described previously
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[29,30].
Accepted Article
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OVA-specific immune responses
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Erythrocyte-depleted mediastinal lymph node (LLN) cells (2x105) from OVA- or PBS-treated
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mice were co-cultured in the presence or absence of 20µg/ml OVA grade VI at 37 °C in RPMI
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1640 medium (PAA, Linz, Austria) containing 5% FCS, penicillin/streptomycin, sodium-
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pyruvate, non-essential amino acids and -mercaptoethanol (all PAA). After 72 hours culture
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supernatants were analysed for cytokine levels by ELISA.
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Cytokine and OVA-specific immunoglobulin determination by ELISA
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To decipher in situ cytokine levels, weighed lung samples were placed in 500µl of RPMI 1640
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medium (without supplements) and homogenized using the T10 basic Ultra-Turrax®
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disperser (IKA, Staufen, Germany). Samples were then centrifuged at 16,000g for 10min
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(4°C) and the resulting supernatant frozen at -20°C. The cytokine content was then
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determined by ELISA. OVA-specific IgE and IgG1 levels were measured in the sera of
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individual mice [30]. In brief, 96-well ELISA plates (Nunc , Langenselbold, Germany) were
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coated overnight (4°C) with either 1µg (for IgE) or 0.1µg (for IgG1) OVA grade V diluted in
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50mM Tris (Roth®, Karlsruhe, Germany) solution containing 3% BSA (PAA; blocking buffer).
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After washing and blocking, sera was diluted in blocking buffer (1:200 to 1:100,000 dilutions)
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and standards of mouse α-OVA IgE or IgG1 antibodies (Biozol, Eching, Germany) were
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applied in two-fold serial dilutions and incubated overnight (4°C). Subsequently, plates were
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washed and α-mouse IgE or IgG1 biotinylated detection antibodies (Biozol) were applied and
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incubated for 2h (RT). After further washing streptavidin-horseradish peroxidase (HRP) 8 This article is protected by copyright. All rights reserved.
conjugate (R&D Systems GmbH, Wiesbaden, Germany) was added and plates were
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incubated for 30min (RT) in the dark. After a final washing step, BD OptEIA™ TMB substrate
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(BD, Heidelberg, Germany) was applied and reactions were stopped with 2M H 2SO4. Finally,
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ODs were determined at 450nm using the SunriseTM ELISA microplate reader (Tecan,
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Crailsheim, Germany). The concentration of the samples was then calculated according to
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the standard curve.
Accepted Article
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Histochemistry and evaluation of lung inflammation
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Paraffin embedded sections (3µm) from the left lungs of individual mice were stained with
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PAS (Periodic acid-Schiff), which allows the detection of goblet cells in lung basilar
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membranes. Sections were analyzed microscopically for tissue inflammation and goblet cell
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hyperplasia according to previously described methods [29,30]. In short, tissue inflammation
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was determined by the degree of visual thickness of the basal membrane which was graded
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on a scale from 0 to 3 (inflammation score). To determine goblet cell hyperplasia, goblet
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cells within the basal membrane were counted and results are presented as the percentage
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of goblet cells per length of basal membrane.
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Dendritic cell generation and in vitro inflammasome assays
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Bone-marrow derived dendritic cells (BMDC) from wildtype C57BL/6, wildtype BALB/c and
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ASC-/- BALB/c mice were generated using 10µg/ml GMCSF (Peprotech, USA) as previously
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described [15]. To assess IL-1 activation, 1x105 BMDC were stimulated in RPMI medium
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containing 10% FCS and supplements (PAA) for 6 hours with LPS (5ng/ml Invivogen, San
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Diego, USA) and thereafter with ATP (5mM, Carl Roth, Karlsruhe, Germany) for 1 hour. IL-1
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in the culture supernatant was measured by ELISA according to manufacturer guidelines 9 This article is protected by copyright. All rights reserved.
(eBiosciences).
Accepted Article
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Statistical analysis
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Statistical differences were analyzed using GraphPad Prism 5 software (San Diego, CA, USA).
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Parametrically distributed data were analyzed using unpaired t-tests or one-way ANOVA.
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Parametric data are represented as mean ± SD.
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10 This article is protected by copyright. All rights reserved.
Accepted Article
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Results
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Lack of NLRP3 inflammasome reduces the development of allergic airway inflammation
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Since the majority of inflammasome activation studies have been performed in C57BL/6
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mice, we first investigated whether bone-marrow derived dendritic cells (BMDCs) generated
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from BALB/c mice were able to produce IL-1in vitro using a standard assay [15]. As shown
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in Supporting information, Fig. S2a, BMDC derived from C57BL/6 mice produced high
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amounts of IL-1 following LPS priming and ATP activation. BALB/c BMDC were also able to
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secrete sufficient amounts of IL-1which were significantly reduced using ASC-deficient
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BMDC, confirming that BALB/c mice possess functional inflammasome activation [31,32]. To
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determine the requirement of the NLRP3 inflammasome on the development of AAI, groups
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of wildtype (WT), ASC- and NLRP3-deficient BALB/c mice were sensitized with PBS or OVA
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(see methods and Supporting information, Fig. S1a). The three rounds of sensitization were
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administered s.c. in the absence of an adjuvant such as aluminium hydroxide (Alum) since it
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is known that Alum can induce the NLRP3 inflammasome and thus IL-1 secretion [33]. In
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addition, Conrad et al. demonstrated that this adjuvant-free OVA model can induce AAI in a
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manner similar to models using adjuvants [29]. In the model applied here, mice were
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challenged with aerosolic OVA over three consecutive days and analyzed 72 hours thereafter
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(Supporting information, Fig. S1a). A primary parameter of AAI is the influx of immune cells
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into the BAL and the induction of asthmatic symptoms can be clearly observed when one
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compares the cellular content in OVA-WT mice to that found in PBS-WT groups (Fig. 1a-c).
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On first glance, levels of leucocyte infiltration were comparable between OVA-treated WT
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and inflammasome-deficient strains (Fig. 1a). However, upon further analysis, both knockout
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strains showed a significant reduction in the percentage of eosinophils (Fig. 1b) and total 11
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numbers of eosinophils were significantly reduced in ASC-deficient mice (Supporting
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information, Fig. S3a). Inflammasome-deficient mice had significantly higher numbers of
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macrophages (Fig. 1c and Supporting information, Fig. S3b). In contrast, no neutrophil
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infiltration could be observed in either wildtype or inflammasome-deficient mice
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(Supporting information, Fig. 3c). In the control groups of mice, no differences were
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observed in the aforementioned parameters (Fig. 1a-c and Supporting information, Fig. S3a-
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c). In terms of AHR, sensitized ASC-/- and NLRP3-/- mice showed a significant reduction in
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airway resistance (Fig. 1d).
Accepted Article
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Lung inflammation and goblet cell hyperplasia are reduced in NLRP3 -/- and ASC-/- mice
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To verify the finding that NLRP3 inflammasome-deficient mice had reduced experimental
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asthma, lung sections were analyzed for the degree of inflammation and goblet cell
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hyperplasia (bright red colouring) [29,30]. When compared to lung sections of PBS-sensitized
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mice (Fig. 2a-c), strong inflammation could be clearly observed in sections from OVA-
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sensitized and challenged WT mice (Fig. 2d) and this was visibly reduced in ASC-/- (Fig. 2e)
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and NLPR3-/- mice (Fig. 2f). To quantify these impressions, the extent of inflammation (Fig.
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2g) and the amount of goblet cell hyperplasia (Fig. 2h) were measured in each individual
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mouse. When compared to the pathology found in groups of OVA-WT mice, both
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parameters were significantly reduced in ASC-/- strains (Fig. 2g,h). NLRP3-/- mice showed a
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significant reduction in the inflammation score (Fig. 2g) but only a mild reduction in the
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percentage of goblet cells (Fig. 2h). These data confirm that the absence of inflammasome
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components dampens OVA-induced AAI.
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NLRP3 inflammasome activation alters TH cytokine responses 12
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OVA-induced allergic airway inflammation is associated with strong OVA-specific TH
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responses. To assess whether the diminished airway responses in inflammasome-deficient
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mice were reflected in their TH profiles, various cytokines were measured in the BAL and
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within cell-culture supernatants from mediastinal lymph node (LLN) cells that were re-
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stimulated ex vivo with OVA. Fig. 3a shows that levels of IL-5 and IL-13 but not IFN- in the
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BAL were significantly down-regulated in both ASC-/- and NLRP3-/- mice. However, OVA
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stimulated cells from the local draining lymph node of inflammasome-deficient mice
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secreted significantly less IFN- but not IL-5, when compared to WT animals. In contrast, IL-
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10 responses were moderately elevated (non-significantly), indicating possible elevations in
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regulatory processes in ASC- and NLRP3-deficient mice (Fig. 3b). IL-10 levels within the BAL
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and IL-13 levels of OVA stimulated lymph node cells were not altered in inflammasome-
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deficient mice (data not shown). Interestingly, no IL-17 responses could be detected within
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the BAL or OVA stimulated lymph node cells (data not shown). Differential regulation was
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not observed in OVA-specific B cell responses since levels of IgE and IgG1 in the sera were
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comparable in wildtype and inflammasome-deficient mice (Fig. 3c).
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Reduced eosinophilia but not lung inflammation and function in the absence of the IL-1R1
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Levels of in situ IL-1, normalised to individual lung weight, were determined in the lungs of
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OVA-exposed WT and inflammasome-deficient mice and surprisingly no differences were
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observed on the final day of analysis (Fig. 4a). However, directly after the last OVA exposure
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(day 28), significantly higher levels of IL-1 were determined in the WT group (Fig. 4b). This
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suggested that IL-1 plays a critical role during the primary inflammation stages of asthma
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but is rapidly surpassed by other immune mechanisms. In contrast, IL-18 levels were slightly
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higher in the WT group on day 28 but significantly higher on day 31 (Supporting information, 13
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Figs. S2b and c, respectively). To further decipher the role of functional IL-1, we studied AAI
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development in mice deficient for the IL-1 receptor type 1 (Fig. 4c-i). Within the BAL, no
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differences were observed between the two groups of mice in terms of total leucocyte
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numbers (Fig. 4c). However, OVA-sensitized IL-1R1-/- mice demonstrated significantly lower
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levels of eosinophils (Fig. 4d and Supporting information, Fig. S3d), whereas macrophage
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numbers were comparable to wildtype mice (Supporting information, Fig. S3e). Again, very
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few neutrophils could be detected in the BAL (Supporting information, Fig. S3f). With regards
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to pathology, IL-1R1-/- mice showed no significant reduction in either inflammation score
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(Fig. 4e) or goblet cell influx (Fig. 4f). Interestingly, PBS-treated IL-1R1-/- mice already
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presented enhanced lung inflammation and goblet cell hyperplasia when compared to naive
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WT mice (Fig. 4e,f). Moreover, when compared to PBS groups of WT mice, the inflammation
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in OVA-treated WT groups was significantly enhanced but this was not the case in IL-1R1-/-
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mice (Fig. 4e). Airway resistance levels in PBS-treated IL-1R1-/- mice were almost equal to
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those resulting from OVA-treated IL-1R1-/- mice and both groups were higher than in OVA-
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treated WT mice (Fig. 4g). Interestingly, levels of IL-18 were equal within BAL of asthmatic
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wildtype and IL-1R1-deficient mice (Supporting information, Fig. S2d). Levels of IL-13 and
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IFN- within the BAL were increased in IL-1R1-deficient mice whereas levels of IL-5 were
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significantly lower (Fig. 4h). As with all other inflammasome-deficient animals, OVA-specific
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immunoglobulin levels remained unchanged (Fig. 4i). These data show that functional IL-1
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does play a role during AAI and appears to be important for the initiation of immune
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responses and maintaining the correct balance of cytokines and the cellular composition
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within the lung.
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Administration of Anakinra during OVA-challenge reduces the development of AAI 14
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Finally, we aimed to decipher whether bio-active IL-1 was critical during the sensitization
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phase or challenge phase of AAI. IL-1 signalling can be blocked through the administration of
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drugs, such as the endogenous antagonist IL-1Ra Anakinra (Kineret®). Indeed the use of such
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drugs has been shown to ameliorate IL-1 mediated diseases such as rheumatoid arthritis or
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type 2 diabetes [1,3]. Thus, BALB/c mice were treated with different doses of Anakinra
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during either the sensitization (AnakinraSens) or challenge (AnakinraChall) phases of AAI (see
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Supporting information, Figs. S1b and c, respectively). Treatment doses of Anakinra were
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either 15 or 100mg/kg i.p. or 150mg/kg s.c. since the administration of Anakinra in patients
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is usually applied in this manner. These data are respectively depicted in Supporting
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information, Figs. S4, S5 and Fig. 5. Interestingly, mice injected with any of the employed
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doses of Anakinra during challenge had significantly reduced eosinophil numbers in the BAL
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but this was not observed in mice treated during sensitization (Fig. 5a and Supporting
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information, Figs. S3g, S4a and S5a). When compared to doses of 15 or 100mg/kg i.p.
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(Supporting information Figures S4b-c and S5b-c), the increased dose of Anakinra via the s.c.
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route led to significantly reduced disease parameters such as lung inflammation and goblet
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cell hyperplasia (Fig. 5b,c). With regards to immune profiles, levels of IL-13 and IFN- but not
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IL-5, were strongly down-regulated in the AnakinraChall group but not the AnakinraSens group
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(Fig. 5d c.f. filled squares and grey triangles). These modulations to OVA-specific responses
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were only partially observed using lower doses of Anakinra (Supporting information, Figs.
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S4d and S5d). Again, levels of OVA-specific IgG1 and IgE were not altered in any of the
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applied experimental scenarios (Fig. 5e and Supporting information, Figs. S4e and S5e).
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Moreover, no differences in macrophage levels and again no neutrophil infiltration could be
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detected (Supporting information, Figs. S3h,i). However, since Anakinra treatment during
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the challenge phase significantly reduced several AAI parameters we also measured the
Accepted Article
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15 This article is protected by copyright. All rights reserved.
effects of Anakinra on airway hyperresponsiveness. Interestingly, when compared to OVA
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groups airway resistance was significantly reduced in Anakinra-treated OVA groups (c.f.
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closed squares and closed triangles in Fig. 5f). Indeed, levels in the Anakinra-treated group
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were comparable to control groups of mice (c.f. PBS (open squares) and OVA+Anakinra
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(closed triangles). Thus, in contrast to the data sets using IL-1R1-deficient mice which
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showed no significant differences in lung pathology and function the data presented here
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reveals that specific blocking of IL-1 signaling during the acute challenge phase leads to
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reduced inflammatory responses against OVA. Moreover, since the PBS IL-1R1 knockout
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group showed elevated AHR and moderately increased inflammation we demonstrate that a
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permanent loss of IL-1 signaling has a fundamental effect at the steady state. In conclusion,
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the data reveal that specific blocking of IL-1 signalling during the acute challenge phase leads
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to reduced inflammatory responses against OVA and opens up an avenue for exploration
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into possible therapeutic strategies.
Accepted Article
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16 This article is protected by copyright. All rights reserved.
Accepted Article
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Discussion
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In vitro, several agents have been shown to trigger the assembly of the NLRP3
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inflammasome and the consequent release of functional IL-1[13,15,19,20]. However, the
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dysregulation of IL-1 can elicit severe consequences, such as the rare inherited
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inflammasome-mediated pathophysiology of autoinflammatory diseases including Muckle-
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Wells Syndrome and Familial cold autoinflammatory syndrome [34,35]. In both of these
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cases, the genetic defect lies in multiple point mutations in the NACHT domain of the NLRP3
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protein which results in a permanent activation of the NLRP3 inflammasome [36,37]. These
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conditions are classified as cryopyrin-associated periodic syndromes (CAPS) which are a
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subset of autoinflammatory disorders [1,33] and are distinguished from autoimmune
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diseases since they fail to induce antigen-specific T cell responses and high antibody titers
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[35]. Research correlating NLRP3 inflammasome activation and the pathogenesis of these
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disorders has exploded over the last decade and further evidence shows a fundamental role
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in the development of other diseases including cancer [1-3,38]. With regards to the latter,
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murine models of chronic pulmonary fibrotic disorders using silica and asbestos have
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correlated IL-1 and the onset of inflammation induced lung cancer [19,39].
361 362
The list of diseases that have been successfully treated with the IL-1Ra Anakinra (Kineret®), a
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non-glycosylated recombinant form of the naturally occurring IL-1Ra, encompasses not only
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autoinflammatory diseases such as Sweet Syndrome [40] and Relapsing Polychondritis [41]
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but illnesses such as gout and type 2 diabetes [1-3]. Within those studies, Anakinra
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demonstrated high efficacy and no adverse side effects. The data presented here display
367
that administration of Anakinra to OVA-allergic mice ameliorates disease parameters, such 17
This article is protected by copyright. All rights reserved.
as eosinophil infiltration and lung inflammation. We blocked IL-1 signalling during either the
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sensitization or challenge phases and determined that blocking IL-1 signalling during the
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challenge phase produced the most beneficial results. In accordance, a recent study
371
demonstrated, that administration of IL1-alpha only during the challenge phase (in contrast
372
to the sensitization phase) had disease aggravating effects [42]. Even though studies have
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demonstrated that the IL-1 family members IL-18 and IL-33 contribute to allergic airway
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inflammation [43,44], Anakinra specifically blocks IL-1 and IL-1 and therefore indicates
375
that the observed effects here are independent of these cytokines. However, further studies
376
should be performed to identify whether simultaneous blocking of several IL-1 family
377
members during the challenge phase, such as IL-18 and IL-33, is more effective. Even though
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Anakinra has a good safety record it requires daily injections due to its short half-life (4-6
379
hours). Alternative agents such as Rilonacept (Regeneron®; IL-1 trap) and Canakinumab
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(Ilaris®; anti-IL-1 antibody), which have much longer half-lives and specifically block IL-1,
381
are currently being assessed in drug trials [45,46]. Thus, it will be interesting to decipher
382
whether these alternative treatments or new upcoming drugs dampen allergic responses in
383
man.
Accepted Article
368
384 385
As mentioned above, the beneficial effects of Anakinra were observed upon application
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during the challenge phase. In the studies comparing inflammasome-deficient and wildtype
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animals, we were initially puzzled that the levels of IL-1 within the lungs were comparable
388
on the final day of analysis (day 31). However, further investigation revealed that in WT
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mice, levels were significantly higher directly after the last exposure to OVA (day 28). These
390
data indicated that the first inflammatory response depended on NLRP3 inflammasome
391
activation that subsequently led to the release of functional IL-1. This hypothesis was 18
This article is protected by copyright. All rights reserved.
verified to some extent using IL-1R1-deficient mice since these mice also displayed reduced
393
eosinophil infiltration and levels of IL-5 in the BAL. However, they also demonstrated
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elevated levels of IL-13 and IFN- implying that additional factors play a role in the induction
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of local TH2 and TH1 responses within the IL-1R1-deficient mice. Another intriguing aspect of
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these studies was observed in naïve IL-1R1 knockout mice since it appeared that a
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continuous loss of functional IL-1 signalling in the steady-state had a more fundamental
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effect since deficient animals showed elevated basal levels of inflammation, goblet cell
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numbers and moreover showed higher airway resistance than OVA-treated wildtype mice.
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Such effects were not observed in PBS-sensitized mice that were treated with Anakinra
401
demonstrating that selective blocking of IL-1 signalling for a certain period of time does not
402
influence cellular composition and immune responses per se. In correlation, TH1 and TH2
403
levels in the BAL were reduced in Anakinra-treated (challenge) and inflammasome-deficient
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asthmatic mice, confirming that NLRP3 inflammasome-mediated IL-1responses are crucial
405
for the induction of local inflammation within the lung. Besides the functional relevance of
406
IL-1, we show here that IL-18 is important during AAI. Indeed, levels of IL-18 were
407
significantly reduced in ASC- and NLPR3-deficient strains whereas levels were comparable
408
between WT and IL-1R1-/- mice. Since IL-18 is an important TH1 inducing proinflammatory
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cytokine [43] this may explain the reduced IFN- levels observed in asthmatic ASC-/- and
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NLPR3-/- mice but not IL-1R1-/- animals. With regards to OVA-specific responses of re-
411
stimulated lung lymph node cells only IFN- responses were dampened in inflammasome-
412
deficient mice and interestingly, IL-10 levels were actually elevated, albeit not significantly,
413
demonstrating that inflammasome activation influences TH immune responses [15]. IL-17
414
responses were also measured in BAL and antigen-specific culture assays but no differences
415
between the groups could be observed and moreover, levels were extremely low in any of
Accepted Article
392
19 This article is protected by copyright. All rights reserved.
the assessed experiments (data not shown). In addition, almost no IL-17A and IL-17F mRNA
417
expression within asthmatic lungs of WT and ASC-deficient mice could be determined using
418
quantitative RT-PCR (data not shown). These results are in contrast to previous findings since
419
studies have reported that alongside other cytokines, IL-1 induces TH17 immune responses
420
[47-49] that can influence allergic asthma development [50,51]. Moreover, several studies
421
also reported that IL-17 favours neutrophil recruitment leading to the induction of
422
neutrophilia and lung inflammation [52,53]. Interestingly, we could hardly detect neutrophils
423
within the BAL and the frequency of those cells was extremely low compared to other
424
studies [22,23,30]. This might be a possible explanation as to why no IL-17 responses could
425
be detected in any of our currently described experiments. However, the lack of neutrophils
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within the lungs of asthmatic BALB/c mice and the total absence of IL-17 responses remains
427
unclear. Further studies should investigate the role of neutrophils in correlation with IL-17
428
responses within allergic airway diseases of BALB/c mice.
Accepted Article
416
429 430
The data presented here demonstrate that NLRP3 inflammasome activation is essential for
431
the initiation of inflammatory responses within the lung and the modulation of TH2 immune
432
responses but is dispensable for immunoglobulin production since no differences were
433
observed in levels of OVA-specific IgG1 and IgE. In contrast, Besnard et al. demonstrated that
434
OVA-specific IgE levels were strictly dependent on NLRP3 inflammasome activation, as well
435
as all the other asthmatic parameters [23]. However, this study was performed with C57BL/6
436
mice, which, in contrast to BALB/c mice develop less pronounced AAI and have dominant
437
TH1 based immune responses [25-28]. Moreover, Allen et al. showed that allergic airway
438
inflammation within NLRP3-/- mice on C57BL/6 background is similar to WT control mice [24].
439
These contrasting findings demonstrate the complexity of allergic asthma development and 20
This article is protected by copyright. All rights reserved.
highlight the necessity to employ further distinct methods to analyse the role of cytokines in
441
addition to complete knockout mouse models. Overall, the mechanisms of inflammasome
442
activation during AAI still remains unclear although the data shown here indicate that the
443
NLRP3 inflammasome may play a larger role in controlling AHR and airway remodeling rather
444
than inflammation in AAI. Nevertheless, it has been reported that ATP levels are increased in
445
asthmatic patients [54] and in the BAL of OVA-treated mice [50]. Interestingly, mice that lack
446
the functional purinergic P2-receptor P2X7 which recognizes extracellular ATP displayed
447
reduced features of acute and chronic asthma [55] and epithelial damage has been shown to
448
release uric acid and ATP which provokes NLRP3 inflammasome activation [49,56,57]. Thus,
449
the possible release of DAMPs (i.e. ATP) during allergen exposure could be responsible for
450
NLRP3 inflammasome activation during AAI. Collectively, the data presented within this work
451
demonstrate that NLRP3 inflammasome activation and IL-1 release play a crucial role in the
452
pathogenesis of experimental allergic asthma. Moreover, we show that administration of
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Anakinra during the acute challenge phase ameliorates allergic airway inflammation and
454
suggests that blocking IL-1 signalling could provide the basis for novel treatment strategies.
Accepted Article
440
455
21 This article is protected by copyright. All rights reserved.
Accepted Article
456 457
Acknowledgements
458
We thank Sabine Paul (MIH) and Vanessa Krupp and Sandra Arriens (IMMIP) for excellent
459
technical support and Thomas Ruppersberg (Philipps-University Marburg, Germany) for
460
helping with AHR measurements. Moreover, we thank Aubry Tardivel (University of
461
Lausanne, Switzerland) for supplying inflammasome deficient mice. In memoriam of Prof.
462
Jürg Tschopp (University of Lausanne, Switzerland). This works was supported by the SFB
463
(Sonderforschungsbereich) Transregio Tr22. The position of MR was funded by the Else
464
Kröner Fresenius Stiftung (EKFS A47/2010). MR, KS, SS, SH, HG and LEL performed the
465
experiments. MR, LEL, DB, HG and CPdC designed the experiments and provided essential
466
expertise. MR, CPdC and LEL wrote the manuscript.
467 468
Conflict of Interest
469
The authors declare no commercial or financial conflict of interest.
470
22 This article is protected by copyright. All rights reserved.
Accepted Article
471 472
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Figure Legends
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Fig.
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hyperresponsiveness. Groups of WT (PBS: n=11; OVA: n=27), ASC-/- (PBS: n=9; OVA: n=15) or
621
NLRP3-/- (PBS: n=6; OVA: n=20) mice were sensitized with either PBS or OVA (days 0, 7, 14)
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and subsequently challenged with OVA (days 26-28) to induce allergic airway inflammation.
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On day 31, (a) total leucocytes and the percentage of (b) eosinophils and (c) macrophages
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were determined within the BAL. (a-c) Symbols show mean ± SD of individual mice from
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three independent experiments and asterisks show significant differences between the
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indicated brackets (***p
2
Received date: 12/17/2013
3
Revised date: 06/04/2014
4
Accepted date: 06/12/2014
5
Title:
Accepted Article
1
6
Functional relevance of NLRP3 inflammasome-mediated IL-1 during acute allergic airway inflammation1
7
Short title:
Anakinra ameliorates allergic airway inflammation
8
Authors:
Manuel Ritter1,2, Kathrin Straubinger2, Stephanie Schmidt2, Dirk H. Busch2,3,
9
Stefanie Hagner4, Holger Garn4, Clarissa Prazeres da Costa2* and Laura E. Layland1,2*
10
1
11
Affiliations:
Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),
12
University Clinic Bonn, Bonn, Germany; 2Institute of Medical Microbiology, Immunology and
13
Hygiene (MIH), Technische Universität München, Munich, Germany; 3Clinical Cooperation
14
Groups ‘‘Antigen-specific Immunotherapy’’ and “Immune Monitoring”, Helmholtz Center
15
Munich (Neuherberg) and Technische Universität München, Munich, Germany; 4Institute of
16
Laboratory Medicine and Pathobiochemistry, Medical Faculty, Philipps-University Marburg,
17
Marburg, Germany.
18 19
*equal contribution
20 21
Corresponding Authors
22
Dr. L. E. Layland
23
Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Clinic
24
Bonn, Sigmund Freud Strasse 25, 53105, Bonn, Germany.
25
Tel: +49 228 287 11387
26
[email protected];
27 28
Dr. C. Prazeres da Costa
29
Institute of Medical Microbiology, Immunology and Hygiene (MIH), Technische Universität
30
München, Trogerstrasse 30, 81675, Munich, Germany; This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cei.12400
1 This article is protected by copyright. All rights reserved.
Tel: +49 89 4140 4130
32
[email protected]
Accepted Article
31
33 34
Keywords
35
Allergy, Anakinra, IL-1, IL-1 receptor antagonist, NLRP3-inflammasome.
36
Abbreviations: AAI: allergic airway inflammation; AHR: Airway hyper-responsiveness; ASC:
37
apoptosis-associated speck-like protein containing a caspase recruitment domain; BAL:
38
bronchoalveloar lavage; GINA: Global Initiative of Asthma; IL-1R: IL-1 receptor; IL-1Ra: IL-1
39
receptor antagonist; NLRP3: nucleotide oligomerization domain (NOD)-like receptor family,
40
pyrin domain 3; OVA: ovalbumin; RT: room temperature.
41 42
2 This article is protected by copyright. All rights reserved.
Accepted Article
43 44
Summary
45
Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However,
46
certain symptomatic individuals remain at risk for serious morbidity and mortality which
47
prompts the identification of novel therapeutic targets and treatment strategies. Thus, using
48
an adjuvant-free TH2 murine model we have deciphered the role of IL-1 signalling during
49
allergic airway inflammation (AAI). Since functional IL-1 depends on inflammasome
50
activation we first studied asthmatic manifestations in specific inflammasome-deficient
51
(NLRP3-/- and ASC-/-) and IL-1 receptor type 1-/- (IL-1R1-/-) mice on the BALB/c background. To
52
verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung
53
pathology, airway hyper-responsiveness and OVA-specific immune responses. In the absence
54
of NLRP3 inflammasome-mediated IL-1 release all symptoms of AAI were reduced except
55
OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling
56
reduced asthmatic development we administered the IL-1R antagonist Anakinra (Kineret®)
57
during critical immunological time-points: sensitization or challenge. Amelioration of
58
asthmatic symptoms was only observed when Anakinra was administered during OVA
59
challenge. Our findings indicate that blocking IL-1 signalling could be a potential
60
complementary therapy for allergic airway inflammation.
61
3 This article is protected by copyright. All rights reserved.
Accepted Article
62 63
Introduction
64
Over the last decade, the importance of inflammasome mediated release of IL-1 has been
65
shown in a variety of models and moreover, has provided the basis for the treatment of
66
several auto-inflammatory diseases such as rheumatoid arthritis, gout and type 2 diabetes
67
[1-3]. In addition, several studies have demonstrated that IL-1 plays an important role
68
during bronchial asthma development. Indeed, asthmatic patients were shown to harbour
69
elevated levels of IL-1 in their bronchoalveloar lavage (BAL) [4]. Other studies have further
70
reported that this proinflammatory cytokine promotes the infiltration of eosinophils and
71
inflammation within the lung and encourages both mast and T cell activation [5,6]. Currently,
72
300 million people are estimated to be affected by asthma worldwide (GINA 2010) and the
73
prevalence continues to increase. Although current therapeutic strategies, such as
74
corticosteroids and long or short acting beta agonists, can control attacks and disease
75
progression, a quarter of a million people still succumbed to the syndrome in 2009 (GINA
76
2009) [7,8]. Asthma is characterized by dominant TH2 immune responses including enhanced
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IL-4, IL-5 and IL-13 responses, allergen-specific immunoglobulin production, eosinophilia,
78
airway inflammation, bronchoconstriction and airway hyperresponsiveness (AHR) [9-11].
79
Thus, when assessing the onset of asthma in murine models is it imperative to address both
80
cellular and pathological aspects.
81 82
The secretion of functional bioactive IL-1 however, depends on the inflammasome which
83
mediates, via caspase-1 activation, the cleavage of the inactive cytokine precursor (pro-IL-
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1 into the active form (IL-1. In general, this multiprotein complex contains proteins such
85
as central oligomerization domain (NACHT), a leucine rich repeat (LRR) domain, apoptosis4 This article is protected by copyright. All rights reserved.
associated speck-like protein containing caspase recruitment domain (ASC) and caspase-1
87
[12-14]. For instance, the well-studied NLRP3 inflammasome plays an essential role in a
88
variety of scenarios and is triggered by numerous pathogens such as parasites [15], fungi
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[16], bacteria [17] or danger signals such as ATP [18] or crystalline silica and asbestos
90
[19,20]. However, its role during allergic asthma remains somewhat controversial. For
91
example, IL-1 receptor type 1 (IL-1R1)-deficient mice present reduced allergic responses and
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lung inflammation [21,22] and ovalbumin (OVA)-treated NLRP3-/- mice exhibit severely
93
dampened lung inflammation, OVA-specific immunoglobulins, cytokine responses and
94
eosinophil infiltration [23]. In contrast to the latter, Allen et al. demonstrated that allergic
95
airway inflammation within NLRP3-deficient mice is similar to wildtype control mice [24].
96
Both of these NLRP3 inflammasome studies were performed using C57BL/6 mice which are
97
renowned for their dominant TH1 based immune responses and reduced allergic airway
98
inflammation when compared to TH2-biased BALB/c mice [25-28]. Therefore, we have
99
assessed the role of the NLRP3 inflammasome and functional IL-1 during OVA-induced AAI
100
in a BALB/c murine model and observed altered antigen-specific TH responses, reduced
101
eosinophil infiltration and airway inflammation in mice deficient for NLRP3, ASC and the IL-
102
1R1. To analyze the dynamics of functional IL-1 in more detail, we blocked IL-1 signalling
103
using the IL-1R antagonist (IL-1Ra) Anakinra. The application of this therapy ameliorated
104
allergic airway inflammation only during the challenge phase and implies that blocking IL-1
105
signalling could complement current asthma treatment regimen.
Accepted Article
86
106
5 This article is protected by copyright. All rights reserved.
Accepted Article
107 108
Materials and Methods
109
Mice
110
BALB/c and C57BL/6 wildtype mice were purchased from Harlan® (Borchen, Germany). ASC-,
111
NLRP3- and IL-1R1-deficient mice on BALB/c background (backcrossed at least nine
112
generations) were a kind gift from Prof. Jürg Tschopp (University of Lausanne, Switzerland).
113
Mice were bred under specific pathogen-free (spf) conditions in the animal facilities at the
114
Institute of Medical Microbiology, Immunology and Hygiene (Munich, Germany) in
115
accordance with national and EU guidelines 86/809. Experimental mice were sex- and age-
116
matched and the study was approved by the Regierung von Oberbayern, Munich, Germany
117
(Animal License number Az. 55.2.1.54-2532-67-12).
118 119
OVA-induced allergic airway inflammation model and Anakinra treatment
120
Female BALB/c wildtype, ASC-, NLRP3- and IL-1R1-deficient mice were thrice sensitized
121
subcutaneously (s.c.) in the neck with 10µg ovalbumin (OVA) grade VI (Sigma-Aldrich,
122
Taufkirchen, Germany) or PBS (control mice) on days 0, 7 and 14 without adjuvant and
123
consecutively challenged by aerosol inhalation on days 26-28 with 10µg OVA grade V (Sigma-
124
Aldrich). On day 31 (3 days after the last OVA challenge) mice were sacrificed and assessed
125
for allergic airway development (Supporting information, Fig. S1a). For IL-1 type cytokine
126
blocking experiments mice were treated with consecutive doses of Anakinra (Kineret®;
127
Biovitrum, Stockholm, Sweden) during OVA-sensitization or challenge. For details see
128
schemes in Supporting information, Fig. S1b,c. Experiments were performed using either
129
15mg/kg (i.p), 100mg/kg (i.p.) or 150mg/kg (s.c.) in the neck per day.
130
6 This article is protected by copyright. All rights reserved.
Accepted Article
131 132
Measurement of airway hyperresponsiveness (AHR)
133
Airway hyperresponsiveness (AHR) to methacholine (Sigma-Aldrich) was determined using
134
the Flexivent system (SCIREQ, Montreal, Canada). Following anaesthesia the trachea was
135
intubated with a 1.2 mm tracheal cannula and the lungs mechanically ventilated at a
136
respiratory frequency of 150 breaths per min, a tidal volume of 10ml/kg and a positive end-
137
expiratory pressure of 3ml H2O. After exposing mice to aerosolized PBS to retrieve the
138
baseline value, bronchoconstriction was induced using increasing concentrations (1, 2.5, 5,
139
10, 25 and 50 mg/ml in PBS) of aerosolized methacholine using an ultrasonic nebulizer.
140
Dynamic resistance was recorded over 1 minute intervals (every 5 secs) after exposure to
141
defined doses of methacholine via a standardized inhalation manoeuvre (SnapShot-150)
142
[29,30].
143 144
Analysis of bronchoalveolar lavage
145
Mice were euthanized with Narcoren® (Merial, Halbergmoos, Germany) and lungs were
146
flushed twice with 1ml PBS containing proteinase inhibitor cocktail tablets (Roche
147
Diagnostics, Mannheim, Germany). The obtained BAL was weighed and centrifuged at 230g
148
for 5min (4°C). Resulting supernatants were frozen at -20°C and the cytokine content
149
determined using commercially available ELISA kits (eBiosciences, Frankfurt, Germany). The
150
cell pellets were resuspended in PBS containing 2% FCS and counted. To analyze the
151
composition of immune cells within the BAL, 150µl of cell suspension was centrifuged on
152
glass slides at 400rpm for 5min using the Shandon Cytospin 3 centrifuge (Thermo Scientific®,
153
Hamburg, Germany). Glass slides were dried overnight at room temperature (RT) and
154
stained using the Diff-Quick staining set (Medion Diagnostics, Langen, Germany) according to 7 This article is protected by copyright. All rights reserved.
manufacturer’s instructions. Cell differentiation was performed as described previously
156
[29,30].
Accepted Article
155
157 158
OVA-specific immune responses
159
Erythrocyte-depleted mediastinal lymph node (LLN) cells (2x105) from OVA- or PBS-treated
160
mice were co-cultured in the presence or absence of 20µg/ml OVA grade VI at 37 °C in RPMI
161
1640 medium (PAA, Linz, Austria) containing 5% FCS, penicillin/streptomycin, sodium-
162
pyruvate, non-essential amino acids and -mercaptoethanol (all PAA). After 72 hours culture
163
supernatants were analysed for cytokine levels by ELISA.
164 165
Cytokine and OVA-specific immunoglobulin determination by ELISA
166
To decipher in situ cytokine levels, weighed lung samples were placed in 500µl of RPMI 1640
167
medium (without supplements) and homogenized using the T10 basic Ultra-Turrax®
168
disperser (IKA, Staufen, Germany). Samples were then centrifuged at 16,000g for 10min
169
(4°C) and the resulting supernatant frozen at -20°C. The cytokine content was then
170
determined by ELISA. OVA-specific IgE and IgG1 levels were measured in the sera of
171
individual mice [30]. In brief, 96-well ELISA plates (Nunc , Langenselbold, Germany) were
172
coated overnight (4°C) with either 1µg (for IgE) or 0.1µg (for IgG1) OVA grade V diluted in
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50mM Tris (Roth®, Karlsruhe, Germany) solution containing 3% BSA (PAA; blocking buffer).
174
After washing and blocking, sera was diluted in blocking buffer (1:200 to 1:100,000 dilutions)
175
and standards of mouse α-OVA IgE or IgG1 antibodies (Biozol, Eching, Germany) were
176
applied in two-fold serial dilutions and incubated overnight (4°C). Subsequently, plates were
177
washed and α-mouse IgE or IgG1 biotinylated detection antibodies (Biozol) were applied and
178
incubated for 2h (RT). After further washing streptavidin-horseradish peroxidase (HRP) 8 This article is protected by copyright. All rights reserved.
conjugate (R&D Systems GmbH, Wiesbaden, Germany) was added and plates were
180
incubated for 30min (RT) in the dark. After a final washing step, BD OptEIA™ TMB substrate
181
(BD, Heidelberg, Germany) was applied and reactions were stopped with 2M H 2SO4. Finally,
182
ODs were determined at 450nm using the SunriseTM ELISA microplate reader (Tecan,
183
Crailsheim, Germany). The concentration of the samples was then calculated according to
184
the standard curve.
Accepted Article
179
185 186
Histochemistry and evaluation of lung inflammation
187
Paraffin embedded sections (3µm) from the left lungs of individual mice were stained with
188
PAS (Periodic acid-Schiff), which allows the detection of goblet cells in lung basilar
189
membranes. Sections were analyzed microscopically for tissue inflammation and goblet cell
190
hyperplasia according to previously described methods [29,30]. In short, tissue inflammation
191
was determined by the degree of visual thickness of the basal membrane which was graded
192
on a scale from 0 to 3 (inflammation score). To determine goblet cell hyperplasia, goblet
193
cells within the basal membrane were counted and results are presented as the percentage
194
of goblet cells per length of basal membrane.
195 196
Dendritic cell generation and in vitro inflammasome assays
197
Bone-marrow derived dendritic cells (BMDC) from wildtype C57BL/6, wildtype BALB/c and
198
ASC-/- BALB/c mice were generated using 10µg/ml GMCSF (Peprotech, USA) as previously
199
described [15]. To assess IL-1 activation, 1x105 BMDC were stimulated in RPMI medium
200
containing 10% FCS and supplements (PAA) for 6 hours with LPS (5ng/ml Invivogen, San
201
Diego, USA) and thereafter with ATP (5mM, Carl Roth, Karlsruhe, Germany) for 1 hour. IL-1
202
in the culture supernatant was measured by ELISA according to manufacturer guidelines 9 This article is protected by copyright. All rights reserved.
(eBiosciences).
Accepted Article
203 204 205
Statistical analysis
206
Statistical differences were analyzed using GraphPad Prism 5 software (San Diego, CA, USA).
207
Parametrically distributed data were analyzed using unpaired t-tests or one-way ANOVA.
208
Parametric data are represented as mean ± SD.
209
10 This article is protected by copyright. All rights reserved.
Accepted Article
210 211
Results
212
Lack of NLRP3 inflammasome reduces the development of allergic airway inflammation
213
Since the majority of inflammasome activation studies have been performed in C57BL/6
214
mice, we first investigated whether bone-marrow derived dendritic cells (BMDCs) generated
215
from BALB/c mice were able to produce IL-1in vitro using a standard assay [15]. As shown
216
in Supporting information, Fig. S2a, BMDC derived from C57BL/6 mice produced high
217
amounts of IL-1 following LPS priming and ATP activation. BALB/c BMDC were also able to
218
secrete sufficient amounts of IL-1which were significantly reduced using ASC-deficient
219
BMDC, confirming that BALB/c mice possess functional inflammasome activation [31,32]. To
220
determine the requirement of the NLRP3 inflammasome on the development of AAI, groups
221
of wildtype (WT), ASC- and NLRP3-deficient BALB/c mice were sensitized with PBS or OVA
222
(see methods and Supporting information, Fig. S1a). The three rounds of sensitization were
223
administered s.c. in the absence of an adjuvant such as aluminium hydroxide (Alum) since it
224
is known that Alum can induce the NLRP3 inflammasome and thus IL-1 secretion [33]. In
225
addition, Conrad et al. demonstrated that this adjuvant-free OVA model can induce AAI in a
226
manner similar to models using adjuvants [29]. In the model applied here, mice were
227
challenged with aerosolic OVA over three consecutive days and analyzed 72 hours thereafter
228
(Supporting information, Fig. S1a). A primary parameter of AAI is the influx of immune cells
229
into the BAL and the induction of asthmatic symptoms can be clearly observed when one
230
compares the cellular content in OVA-WT mice to that found in PBS-WT groups (Fig. 1a-c).
231
On first glance, levels of leucocyte infiltration were comparable between OVA-treated WT
232
and inflammasome-deficient strains (Fig. 1a). However, upon further analysis, both knockout
233
strains showed a significant reduction in the percentage of eosinophils (Fig. 1b) and total 11
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numbers of eosinophils were significantly reduced in ASC-deficient mice (Supporting
235
information, Fig. S3a). Inflammasome-deficient mice had significantly higher numbers of
236
macrophages (Fig. 1c and Supporting information, Fig. S3b). In contrast, no neutrophil
237
infiltration could be observed in either wildtype or inflammasome-deficient mice
238
(Supporting information, Fig. 3c). In the control groups of mice, no differences were
239
observed in the aforementioned parameters (Fig. 1a-c and Supporting information, Fig. S3a-
240
c). In terms of AHR, sensitized ASC-/- and NLRP3-/- mice showed a significant reduction in
241
airway resistance (Fig. 1d).
Accepted Article
234
242 243
Lung inflammation and goblet cell hyperplasia are reduced in NLRP3 -/- and ASC-/- mice
244
To verify the finding that NLRP3 inflammasome-deficient mice had reduced experimental
245
asthma, lung sections were analyzed for the degree of inflammation and goblet cell
246
hyperplasia (bright red colouring) [29,30]. When compared to lung sections of PBS-sensitized
247
mice (Fig. 2a-c), strong inflammation could be clearly observed in sections from OVA-
248
sensitized and challenged WT mice (Fig. 2d) and this was visibly reduced in ASC-/- (Fig. 2e)
249
and NLPR3-/- mice (Fig. 2f). To quantify these impressions, the extent of inflammation (Fig.
250
2g) and the amount of goblet cell hyperplasia (Fig. 2h) were measured in each individual
251
mouse. When compared to the pathology found in groups of OVA-WT mice, both
252
parameters were significantly reduced in ASC-/- strains (Fig. 2g,h). NLRP3-/- mice showed a
253
significant reduction in the inflammation score (Fig. 2g) but only a mild reduction in the
254
percentage of goblet cells (Fig. 2h). These data confirm that the absence of inflammasome
255
components dampens OVA-induced AAI.
256 257
NLRP3 inflammasome activation alters TH cytokine responses 12
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OVA-induced allergic airway inflammation is associated with strong OVA-specific TH
259
responses. To assess whether the diminished airway responses in inflammasome-deficient
260
mice were reflected in their TH profiles, various cytokines were measured in the BAL and
261
within cell-culture supernatants from mediastinal lymph node (LLN) cells that were re-
262
stimulated ex vivo with OVA. Fig. 3a shows that levels of IL-5 and IL-13 but not IFN- in the
263
BAL were significantly down-regulated in both ASC-/- and NLRP3-/- mice. However, OVA
264
stimulated cells from the local draining lymph node of inflammasome-deficient mice
265
secreted significantly less IFN- but not IL-5, when compared to WT animals. In contrast, IL-
266
10 responses were moderately elevated (non-significantly), indicating possible elevations in
267
regulatory processes in ASC- and NLRP3-deficient mice (Fig. 3b). IL-10 levels within the BAL
268
and IL-13 levels of OVA stimulated lymph node cells were not altered in inflammasome-
269
deficient mice (data not shown). Interestingly, no IL-17 responses could be detected within
270
the BAL or OVA stimulated lymph node cells (data not shown). Differential regulation was
271
not observed in OVA-specific B cell responses since levels of IgE and IgG1 in the sera were
272
comparable in wildtype and inflammasome-deficient mice (Fig. 3c).
Accepted Article
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273 274
Reduced eosinophilia but not lung inflammation and function in the absence of the IL-1R1
275
Levels of in situ IL-1, normalised to individual lung weight, were determined in the lungs of
276
OVA-exposed WT and inflammasome-deficient mice and surprisingly no differences were
277
observed on the final day of analysis (Fig. 4a). However, directly after the last OVA exposure
278
(day 28), significantly higher levels of IL-1 were determined in the WT group (Fig. 4b). This
279
suggested that IL-1 plays a critical role during the primary inflammation stages of asthma
280
but is rapidly surpassed by other immune mechanisms. In contrast, IL-18 levels were slightly
281
higher in the WT group on day 28 but significantly higher on day 31 (Supporting information, 13
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Figs. S2b and c, respectively). To further decipher the role of functional IL-1, we studied AAI
283
development in mice deficient for the IL-1 receptor type 1 (Fig. 4c-i). Within the BAL, no
284
differences were observed between the two groups of mice in terms of total leucocyte
285
numbers (Fig. 4c). However, OVA-sensitized IL-1R1-/- mice demonstrated significantly lower
286
levels of eosinophils (Fig. 4d and Supporting information, Fig. S3d), whereas macrophage
287
numbers were comparable to wildtype mice (Supporting information, Fig. S3e). Again, very
288
few neutrophils could be detected in the BAL (Supporting information, Fig. S3f). With regards
289
to pathology, IL-1R1-/- mice showed no significant reduction in either inflammation score
290
(Fig. 4e) or goblet cell influx (Fig. 4f). Interestingly, PBS-treated IL-1R1-/- mice already
291
presented enhanced lung inflammation and goblet cell hyperplasia when compared to naive
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WT mice (Fig. 4e,f). Moreover, when compared to PBS groups of WT mice, the inflammation
293
in OVA-treated WT groups was significantly enhanced but this was not the case in IL-1R1-/-
294
mice (Fig. 4e). Airway resistance levels in PBS-treated IL-1R1-/- mice were almost equal to
295
those resulting from OVA-treated IL-1R1-/- mice and both groups were higher than in OVA-
296
treated WT mice (Fig. 4g). Interestingly, levels of IL-18 were equal within BAL of asthmatic
297
wildtype and IL-1R1-deficient mice (Supporting information, Fig. S2d). Levels of IL-13 and
298
IFN- within the BAL were increased in IL-1R1-deficient mice whereas levels of IL-5 were
299
significantly lower (Fig. 4h). As with all other inflammasome-deficient animals, OVA-specific
300
immunoglobulin levels remained unchanged (Fig. 4i). These data show that functional IL-1
301
does play a role during AAI and appears to be important for the initiation of immune
302
responses and maintaining the correct balance of cytokines and the cellular composition
303
within the lung.
Accepted Article
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304 305
Administration of Anakinra during OVA-challenge reduces the development of AAI 14
This article is protected by copyright. All rights reserved.
Finally, we aimed to decipher whether bio-active IL-1 was critical during the sensitization
307
phase or challenge phase of AAI. IL-1 signalling can be blocked through the administration of
308
drugs, such as the endogenous antagonist IL-1Ra Anakinra (Kineret®). Indeed the use of such
309
drugs has been shown to ameliorate IL-1 mediated diseases such as rheumatoid arthritis or
310
type 2 diabetes [1,3]. Thus, BALB/c mice were treated with different doses of Anakinra
311
during either the sensitization (AnakinraSens) or challenge (AnakinraChall) phases of AAI (see
312
Supporting information, Figs. S1b and c, respectively). Treatment doses of Anakinra were
313
either 15 or 100mg/kg i.p. or 150mg/kg s.c. since the administration of Anakinra in patients
314
is usually applied in this manner. These data are respectively depicted in Supporting
315
information, Figs. S4, S5 and Fig. 5. Interestingly, mice injected with any of the employed
316
doses of Anakinra during challenge had significantly reduced eosinophil numbers in the BAL
317
but this was not observed in mice treated during sensitization (Fig. 5a and Supporting
318
information, Figs. S3g, S4a and S5a). When compared to doses of 15 or 100mg/kg i.p.
319
(Supporting information Figures S4b-c and S5b-c), the increased dose of Anakinra via the s.c.
320
route led to significantly reduced disease parameters such as lung inflammation and goblet
321
cell hyperplasia (Fig. 5b,c). With regards to immune profiles, levels of IL-13 and IFN- but not
322
IL-5, were strongly down-regulated in the AnakinraChall group but not the AnakinraSens group
323
(Fig. 5d c.f. filled squares and grey triangles). These modulations to OVA-specific responses
324
were only partially observed using lower doses of Anakinra (Supporting information, Figs.
325
S4d and S5d). Again, levels of OVA-specific IgG1 and IgE were not altered in any of the
326
applied experimental scenarios (Fig. 5e and Supporting information, Figs. S4e and S5e).
327
Moreover, no differences in macrophage levels and again no neutrophil infiltration could be
328
detected (Supporting information, Figs. S3h,i). However, since Anakinra treatment during
329
the challenge phase significantly reduced several AAI parameters we also measured the
Accepted Article
306
15 This article is protected by copyright. All rights reserved.
effects of Anakinra on airway hyperresponsiveness. Interestingly, when compared to OVA
331
groups airway resistance was significantly reduced in Anakinra-treated OVA groups (c.f.
332
closed squares and closed triangles in Fig. 5f). Indeed, levels in the Anakinra-treated group
333
were comparable to control groups of mice (c.f. PBS (open squares) and OVA+Anakinra
334
(closed triangles). Thus, in contrast to the data sets using IL-1R1-deficient mice which
335
showed no significant differences in lung pathology and function the data presented here
336
reveals that specific blocking of IL-1 signaling during the acute challenge phase leads to
337
reduced inflammatory responses against OVA. Moreover, since the PBS IL-1R1 knockout
338
group showed elevated AHR and moderately increased inflammation we demonstrate that a
339
permanent loss of IL-1 signaling has a fundamental effect at the steady state. In conclusion,
340
the data reveal that specific blocking of IL-1 signalling during the acute challenge phase leads
341
to reduced inflammatory responses against OVA and opens up an avenue for exploration
342
into possible therapeutic strategies.
Accepted Article
330
343
16 This article is protected by copyright. All rights reserved.
Accepted Article
344 345
Discussion
346
In vitro, several agents have been shown to trigger the assembly of the NLRP3
347
inflammasome and the consequent release of functional IL-1[13,15,19,20]. However, the
348
dysregulation of IL-1 can elicit severe consequences, such as the rare inherited
349
inflammasome-mediated pathophysiology of autoinflammatory diseases including Muckle-
350
Wells Syndrome and Familial cold autoinflammatory syndrome [34,35]. In both of these
351
cases, the genetic defect lies in multiple point mutations in the NACHT domain of the NLRP3
352
protein which results in a permanent activation of the NLRP3 inflammasome [36,37]. These
353
conditions are classified as cryopyrin-associated periodic syndromes (CAPS) which are a
354
subset of autoinflammatory disorders [1,33] and are distinguished from autoimmune
355
diseases since they fail to induce antigen-specific T cell responses and high antibody titers
356
[35]. Research correlating NLRP3 inflammasome activation and the pathogenesis of these
357
disorders has exploded over the last decade and further evidence shows a fundamental role
358
in the development of other diseases including cancer [1-3,38]. With regards to the latter,
359
murine models of chronic pulmonary fibrotic disorders using silica and asbestos have
360
correlated IL-1 and the onset of inflammation induced lung cancer [19,39].
361 362
The list of diseases that have been successfully treated with the IL-1Ra Anakinra (Kineret®), a
363
non-glycosylated recombinant form of the naturally occurring IL-1Ra, encompasses not only
364
autoinflammatory diseases such as Sweet Syndrome [40] and Relapsing Polychondritis [41]
365
but illnesses such as gout and type 2 diabetes [1-3]. Within those studies, Anakinra
366
demonstrated high efficacy and no adverse side effects. The data presented here display
367
that administration of Anakinra to OVA-allergic mice ameliorates disease parameters, such 17
This article is protected by copyright. All rights reserved.
as eosinophil infiltration and lung inflammation. We blocked IL-1 signalling during either the
369
sensitization or challenge phases and determined that blocking IL-1 signalling during the
370
challenge phase produced the most beneficial results. In accordance, a recent study
371
demonstrated, that administration of IL1-alpha only during the challenge phase (in contrast
372
to the sensitization phase) had disease aggravating effects [42]. Even though studies have
373
demonstrated that the IL-1 family members IL-18 and IL-33 contribute to allergic airway
374
inflammation [43,44], Anakinra specifically blocks IL-1 and IL-1 and therefore indicates
375
that the observed effects here are independent of these cytokines. However, further studies
376
should be performed to identify whether simultaneous blocking of several IL-1 family
377
members during the challenge phase, such as IL-18 and IL-33, is more effective. Even though
378
Anakinra has a good safety record it requires daily injections due to its short half-life (4-6
379
hours). Alternative agents such as Rilonacept (Regeneron®; IL-1 trap) and Canakinumab
380
(Ilaris®; anti-IL-1 antibody), which have much longer half-lives and specifically block IL-1,
381
are currently being assessed in drug trials [45,46]. Thus, it will be interesting to decipher
382
whether these alternative treatments or new upcoming drugs dampen allergic responses in
383
man.
Accepted Article
368
384 385
As mentioned above, the beneficial effects of Anakinra were observed upon application
386
during the challenge phase. In the studies comparing inflammasome-deficient and wildtype
387
animals, we were initially puzzled that the levels of IL-1 within the lungs were comparable
388
on the final day of analysis (day 31). However, further investigation revealed that in WT
389
mice, levels were significantly higher directly after the last exposure to OVA (day 28). These
390
data indicated that the first inflammatory response depended on NLRP3 inflammasome
391
activation that subsequently led to the release of functional IL-1. This hypothesis was 18
This article is protected by copyright. All rights reserved.
verified to some extent using IL-1R1-deficient mice since these mice also displayed reduced
393
eosinophil infiltration and levels of IL-5 in the BAL. However, they also demonstrated
394
elevated levels of IL-13 and IFN- implying that additional factors play a role in the induction
395
of local TH2 and TH1 responses within the IL-1R1-deficient mice. Another intriguing aspect of
396
these studies was observed in naïve IL-1R1 knockout mice since it appeared that a
397
continuous loss of functional IL-1 signalling in the steady-state had a more fundamental
398
effect since deficient animals showed elevated basal levels of inflammation, goblet cell
399
numbers and moreover showed higher airway resistance than OVA-treated wildtype mice.
400
Such effects were not observed in PBS-sensitized mice that were treated with Anakinra
401
demonstrating that selective blocking of IL-1 signalling for a certain period of time does not
402
influence cellular composition and immune responses per se. In correlation, TH1 and TH2
403
levels in the BAL were reduced in Anakinra-treated (challenge) and inflammasome-deficient
404
asthmatic mice, confirming that NLRP3 inflammasome-mediated IL-1responses are crucial
405
for the induction of local inflammation within the lung. Besides the functional relevance of
406
IL-1, we show here that IL-18 is important during AAI. Indeed, levels of IL-18 were
407
significantly reduced in ASC- and NLPR3-deficient strains whereas levels were comparable
408
between WT and IL-1R1-/- mice. Since IL-18 is an important TH1 inducing proinflammatory
409
cytokine [43] this may explain the reduced IFN- levels observed in asthmatic ASC-/- and
410
NLPR3-/- mice but not IL-1R1-/- animals. With regards to OVA-specific responses of re-
411
stimulated lung lymph node cells only IFN- responses were dampened in inflammasome-
412
deficient mice and interestingly, IL-10 levels were actually elevated, albeit not significantly,
413
demonstrating that inflammasome activation influences TH immune responses [15]. IL-17
414
responses were also measured in BAL and antigen-specific culture assays but no differences
415
between the groups could be observed and moreover, levels were extremely low in any of
Accepted Article
392
19 This article is protected by copyright. All rights reserved.
the assessed experiments (data not shown). In addition, almost no IL-17A and IL-17F mRNA
417
expression within asthmatic lungs of WT and ASC-deficient mice could be determined using
418
quantitative RT-PCR (data not shown). These results are in contrast to previous findings since
419
studies have reported that alongside other cytokines, IL-1 induces TH17 immune responses
420
[47-49] that can influence allergic asthma development [50,51]. Moreover, several studies
421
also reported that IL-17 favours neutrophil recruitment leading to the induction of
422
neutrophilia and lung inflammation [52,53]. Interestingly, we could hardly detect neutrophils
423
within the BAL and the frequency of those cells was extremely low compared to other
424
studies [22,23,30]. This might be a possible explanation as to why no IL-17 responses could
425
be detected in any of our currently described experiments. However, the lack of neutrophils
426
within the lungs of asthmatic BALB/c mice and the total absence of IL-17 responses remains
427
unclear. Further studies should investigate the role of neutrophils in correlation with IL-17
428
responses within allergic airway diseases of BALB/c mice.
Accepted Article
416
429 430
The data presented here demonstrate that NLRP3 inflammasome activation is essential for
431
the initiation of inflammatory responses within the lung and the modulation of TH2 immune
432
responses but is dispensable for immunoglobulin production since no differences were
433
observed in levels of OVA-specific IgG1 and IgE. In contrast, Besnard et al. demonstrated that
434
OVA-specific IgE levels were strictly dependent on NLRP3 inflammasome activation, as well
435
as all the other asthmatic parameters [23]. However, this study was performed with C57BL/6
436
mice, which, in contrast to BALB/c mice develop less pronounced AAI and have dominant
437
TH1 based immune responses [25-28]. Moreover, Allen et al. showed that allergic airway
438
inflammation within NLRP3-/- mice on C57BL/6 background is similar to WT control mice [24].
439
These contrasting findings demonstrate the complexity of allergic asthma development and 20
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highlight the necessity to employ further distinct methods to analyse the role of cytokines in
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addition to complete knockout mouse models. Overall, the mechanisms of inflammasome
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activation during AAI still remains unclear although the data shown here indicate that the
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NLRP3 inflammasome may play a larger role in controlling AHR and airway remodeling rather
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than inflammation in AAI. Nevertheless, it has been reported that ATP levels are increased in
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asthmatic patients [54] and in the BAL of OVA-treated mice [50]. Interestingly, mice that lack
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the functional purinergic P2-receptor P2X7 which recognizes extracellular ATP displayed
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reduced features of acute and chronic asthma [55] and epithelial damage has been shown to
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release uric acid and ATP which provokes NLRP3 inflammasome activation [49,56,57]. Thus,
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the possible release of DAMPs (i.e. ATP) during allergen exposure could be responsible for
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NLRP3 inflammasome activation during AAI. Collectively, the data presented within this work
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demonstrate that NLRP3 inflammasome activation and IL-1 release play a crucial role in the
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pathogenesis of experimental allergic asthma. Moreover, we show that administration of
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Anakinra during the acute challenge phase ameliorates allergic airway inflammation and
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suggests that blocking IL-1 signalling could provide the basis for novel treatment strategies.
Accepted Article
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21 This article is protected by copyright. All rights reserved.
Accepted Article
456 457
Acknowledgements
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We thank Sabine Paul (MIH) and Vanessa Krupp and Sandra Arriens (IMMIP) for excellent
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technical support and Thomas Ruppersberg (Philipps-University Marburg, Germany) for
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helping with AHR measurements. Moreover, we thank Aubry Tardivel (University of
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Lausanne, Switzerland) for supplying inflammasome deficient mice. In memoriam of Prof.
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Jürg Tschopp (University of Lausanne, Switzerland). This works was supported by the SFB
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(Sonderforschungsbereich) Transregio Tr22. The position of MR was funded by the Else
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Kröner Fresenius Stiftung (EKFS A47/2010). MR, KS, SS, SH, HG and LEL performed the
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experiments. MR, LEL, DB, HG and CPdC designed the experiments and provided essential
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expertise. MR, CPdC and LEL wrote the manuscript.
467 468
Conflict of Interest
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The authors declare no commercial or financial conflict of interest.
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22 This article is protected by copyright. All rights reserved.
Accepted Article
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Figure Legends
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Fig.
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hyperresponsiveness. Groups of WT (PBS: n=11; OVA: n=27), ASC-/- (PBS: n=9; OVA: n=15) or
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NLRP3-/- (PBS: n=6; OVA: n=20) mice were sensitized with either PBS or OVA (days 0, 7, 14)
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and subsequently challenged with OVA (days 26-28) to induce allergic airway inflammation.
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On day 31, (a) total leucocytes and the percentage of (b) eosinophils and (c) macrophages
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were determined within the BAL. (a-c) Symbols show mean ± SD of individual mice from
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three independent experiments and asterisks show significant differences between the
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indicated brackets (***p
