Preventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model
- Matthias Kroker†1,
- Ulrich Sydlik†1,
- Andrea Autengruber1,
- Christian Cavelius2,
- Heike Weighardt3,
- Annette Kraegeloh2 and
- Klaus Unfried1Email author
© Kroker et al. 2015
Received: 18 January 2015
Accepted: 15 June 2015
Published: 4 July 2015
Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation.
Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells.
Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals.
Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization.
Epidemiological studies demonstrated correlations between exposure to particulate air pollution and the incidence and severity of allergic diseases of the airways . Besides sources like environmental tobacco smoke, ultrafine particles from traffic related air pollution have been suggested as modulators of allergic reactions leading to adverse health effects [2, 3]. With the increasing use of nanomaterials in daily life products, human airways might be exposed to inhalable poorly soluble nanoparticles including combustion derived carbon nanoparticles from toners and printers . Carbonaceous nanoparticles in the airways can contribute to both, the exacerbation of the immune response in antigen-exposed sensitized individuals, but also the allergic sensitization against daily life antigens [5, 6].
Mechanistic animal studies demonstrated that the exposure to different kinds of allergens in the presence of diesel exhaust particles (DEP) leads to a potentiation of the antigen-specific sensitization [7, 8]. Comparing chemically different kinds of diesel samples revealed that the amount of organic carbon compounds contaminating the elementary carbonaceous core significantly modulates the strength of this adjuvant effect on the sensitization against antigens . A number of studies however also demonstrate that ultrafine engineered carbon nanoparticles (CNP) which contain only traces of organic carbon enhance the sensitization . In these studies, the increase of antigen-specific IgE was associated with lung inflammation induced by CNP when applied to the airways of the animals . The comparison of chemically similar particle samples of different size classes demonstrate an increasing effectivity associated with the decrease in primary particle size [12, 13]. The influence of spark-generated carbon nanoparticles on immune reactions of the airways has been shown at the level of the immune response after challenging sensitized mice . In a recent study this exacerbating effect was investigated in asthmatic volunteers who were exposed to CNP or filtered air in a cross over design prior to allergen challenge. Interestingly, a delayed effect of CNP on the strength of allergic airway inflammation was observed . In a murine model, the occurrence of markers of oxidative stress was associated with the exacerbation of the allergic lung inflammation by the inhaled CNP . The enhancement of the antioxidant capacity of the airways by adding N-acetyl cysteine led to a significant reduction of the exacerbating effect of the inhaled CNP. Taken together, these studies indicate that the enhanced inflammatory response of the airways as well as the oxidative burst in response to the inhalation of ultrafine particles might be a central mechanism of adverse effects of inhaled nanoparticles on immune diseases of the airways. Strategies which reduce airway inflammation should therefore be able to prevent both the exacerbation of the immune response in sensitized individuals but also the adjuvant effect during allergic sensitization.
We recently described the potential of a group of substances, named compatible solutes, to prevent CNP-induced pro-inflammatory reactions in the airways of exposed animals . These substances can be isolated from extremophilic bacteria which produce them in order to stabilize cellular functions under extreme life conditions like heat or osmotic stress. Based on the principle of preferential exclusion, compatible solutes stabilize macromolecules like membrane lipids and proteins [18–20]. In lung epithelial cells, we were able to demonstrate that CNP-triggered non-canonical activation of EGFR signalling is prevented in the presence of ectoine due to a stabilization of the receptor in the lipid raft signalling compartment of the cells . Addressing inflammatory kinetics in the airways in vivo, we observed similar effects of CNP and of ectoine on cell stress reactions executed on neutrophilic granulocytes (PMN). CNP contribute to the aggravation of the inflammation by suppressing natural apoptosis rates via membrane dependent signalling in both human peripheral PMN and in vivo in exposed rat lungs . The preventive application of ectoine restored apoptosis rates and led to an accelerated resolution of the neutrophilic lung inflammation. As a proof of the biophysical principle of preferential exclusion, we observed that besides ectoine which is isolated from halophilic bacteria, firoin a substance produced by thermophilic bacteria, is also able to prevent the adverse cell stress reactions in lung epithelium and in neutrophils .
The possibility to prevent CNP-induced neutrophilic lung inflammation directly at the level of PMNs offers the possibility to test whether this early reaction of the airways contributes to the development of responses of the adaptive immune system after allergen challenge. In a recent report we described that the aggravation which is mediated by CNP applied during antigen challenge in sensitized mice can be significantly reduced in the presence of ectoine . The current study aimed to investigate how the adjuvant effect of CNP during sensitization against ovalbumin is mediated and whether it is reduced when neutrophilic lung inflammation is attenuated by the application of the compatible solute ectoine. For that purpose, we chose the experimental system of pharyngeal aspiration in which the strength of neutrophilic lung inflammation can be strictly controlled by ectoine. Sensitization via the lower airways and adaptive immune responses after sensitization and challenge were investigated using a mouse model of allergic airway disease in Balb/c mice. In order to separate effects of neutrophilic lung inflammation from direct effects of particles and ectoine on antigen presenting cells, mechanistic studies employing ex vivo differentiated dendritic cells were performed.
Results and discussion
As a pre-requisite for in vivo and in vitro investigations with CNP, particle suspensions were analysed for their physico chemical characteristics. CNP (14 nm, primary diameter) are known to form agglomerates in physiological aqueous solution. Characteristics of the particle suspensions are given in the supplementary data (Additional file 1: Figure S1 and Table S1). In earlier studies we applied such agglomerated nanoparticles in vitro and in vivo and observed size or surface specific effects when compared to bigger non-nano particles which showed very similar agglomerate size distributions .
Taken together, the data of this study demonstrate the importance of enhanced neutrophilic lung inflammation induced by CNP during allergic sensitization as a promoter of allergy. This is demonstrated at the level of antigen-specific IgE, challenge-induced lung inflammation, and TH2 specific changes in the draining lymph nodes. As a possible cause for these effects, the enhanced migration rate of antigen presenting dendritic cells to the lymph node in response to the ongoing inflammation is indicated. The intervention approach using ectoine which moderately but specifically prevents neutrophilic lung inflammation induced by CNP demonstrates the causal connection of particle-induced inflammation and the aggravation of the allergic reaction in this model. This approach, therefore, also offers the possibility to interfere with environmentally-induced immune diseases of the airways like asthma. Ectoine and other compatible solutes have been tested to be highly compatible to mammals. First human studies describing the feasibility and efficacy of ectoine application in upper airways and on the skin suggest to test such compounds as a preventive strategy against adverse health effects in the airways induced by air pollution [30, 31]. Such a strategy might be of relevance for pre-disposed individuals particularly in situations in which exposure of the airways cannot be avoided. This approach is not only relevant for the prevention of environmentally induced effects on the immune system, since engineered nanoparticles have also been described to aggravate airway responses in experimentally induced asthma [32, 33]. Together with the finding that ectoine also reduces the particle-dependent aggravation of the allergic immune reaction, the current study suggests to test this substance in humans to prevent particle induced adverse effects on adaptive immune reactions of the airways.
Carbon nanoparticles (CNP, Printex 90) were obtained from Degussa (Essen, Germany). Stock suspensions (1 mg/ml) of particles were prepared in phosphate buffered saline (PBS) as vehicle by sonication for 60 min. Particles and particle suspensions were characterized as described in the supplementary.
Application via pharyngeal aspiration and bronchoalveolar lavage
Female Balb/cJRj mice (8 weeks old, Janvier, France) were treated with particle suspensions (2.5 mg/kg bodyweight) with or without 1 mM ectoine (Sigma-Aldrich Chemie, Deisenhofen, Germany) or PBS as control solution via pharyngeal aspiration with a volume of 50 μl, under inhalation anaesthesia (isoflurane, 5 % in synthetic air, 2 min). Animals were sensitized by repetitive pharyngeal aspiration with a volume of 50 μl of 1 mg/ml Grade VI Ovalbumin (OVA) (Sigma-Aldrich Chemie, Deisenhofen, Germany) dissolved in PBS. At the indicated time points, mice were challenged by aerosol inhalation (1 % OVA in PBS) for 30 min using a Pari-Boy Nebuliser (Pari, Starnberg, Germany). For labelling of antigen transporting cells, 50 μl of 1 mg/ml Ovalbumin, Alexa Fluor 488 conjugate (OVA-488) (Invitrogen, Carlsbad, CA, USA) was applied via pharyngeal aspiration. Animals were sacrificed by exsanguinations under deep anaesthesia after the indicated exposure times. Bronchoalveolar lavage (BAL) was taken using 4 × 1 ml PBS. The determination of inflammatory cells in BAL by flow cytometry was performed according to the method described by de Haar and colleagues, staining for GR-1 and CD11c . The validity of this method was verified by additional staining procedures for neutrophils (GR-1, CD11b) and macrophages (F4/80, CD11b), for selected samples. All animal experiments were performed after relevant permission according to German animal protection laws.
Blood was drawn from mice on days 21 and 35 by facial vein puncture. Serum was stored at −80 °C until measurement of OVA-specific IgE antibodies by ELISA (MD bioproducts, St Paul, MN) according to the manufacturer’s instructions.
BAL-cells were isolated and stained for flow cytometry. Cell free lavage fluids were subjected to solid-phase ELISA in order to determine CXCL1, IL-4 and IL-13 (R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.
Peribronchial lymph node analysis
Peribronchial lymph nodes were isolated immediately after BAL. After 15 min digestion with 1 mg/ml Collagenase D and 300 U/ml DNase I (both Roche, Mannheim, Germany) in PBS at 37 °C single-cell suspensions were made by using a 100 μm cell strainer (BD Bioscience, Franklyn Lake, NJ, USA). Cell suspensions were incubated for 10 min with red blood cell lysis buffer at 4 C. Then cells were counted and stained for flow cytometry using specific antibodies. Cell suspension were cultured in RPMI-1640 medium with 10 % FCS, 1 % L-Glutamine 2 % Pen/Strep (all obtained from Sigma Aldrich) in round bottom 96-well plate (2 × 105 cells per animal/well) and re-stimulated with 100 mg/ml OVA for 4 days at 37 °C, 5 % CO2. Levels of IL-4 and IL-13 in culture supernatants were measured by ELISA (R&D systems, Minneapolis, MN) according to the manufacturer’s protocol.
Flow cytometry was performed with a FACScanto II Flow Cytometer (BD Bioscience, BD Bioscience, Franklyn Lake, NJ, USA) and analysed with FlowJo 7.6.5. Fluorescently labelled CD11c (N418), GR-1 (RB6-8C5), MHCII (M5/144.15.2), CD4 (GK1.5), CD8b (H35-17.2), CD19 (MB19-1), CCR7 (4B12), F4/80 (BM8) and biotin anti-APC (APC003) were used in various combinations. Antibodies used were from BioLegend (San Diego, CA, USA), except for CD19 (eBioscience, San Diego, CA, USA), CD4 and CD8 (BD Pharmingen). Dendritic cells and macrophages loaded with OVA-488 were also detected by using flow cytometry.
In vitro bone marrow-derived dendritic cell exposures
Bone marrow–derived dendritic cells (BMDCs) were cultured from bone marrow by using recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) (PreproTech, Hamburg, Germany). On day 6, cells were harvested and cell purity was analysed by flow cytometry. Cells had an average purity of 70 %. A stock solution/suspension of CNP or lipopolysaccharides (LPS, Escherichia coli 0111:B4; Sigma-Aldrich) was prepared in PBS. Cells were exposed then for 18 h to CNP (0, 3; 3; 30 mg/ml) with or without 1 mM ectoine, LPS (100 ng/ml) with or without 1 mM ectoine, PBS or ectoine alone, cell free BAL obtained from animals which were treated 12 h with CNP alone or in combination with 1 mM ectoine, and cell free BAL obtained from animals which were treated with CNP alone plus 1 mM ectoine in PBS in vitro. Cell viability was tested by trypan blue (Sigma Aldrich) exclusion test. BMDCs were isolated and stained for flow cytometry for expression of CCR7.
Statistical calculations were performed using IBM SPSS statistics 22. Significant values were calculated either by ANOVA analyses with Tukey’s HSD post hoc testing or by comparison of individual groups by Mann–Whitney U-test. Mean values with standard errors are given. Power calculations for the design of animal experiments were performed using G*Power version 3.1.5 (University of Kiel, Germany). Graphs display means and standard errors.
The authors wish to thank the recent and former members of the groups of Prof. Irmgard Förster and Prof. Charlotte Esser for their assistance in flow cytometric and immunological methods. The technical assistance of Winfried Brock, Ragnhild Wirth, and Petra Groß is gratefully acknowledged. The research was funded by a research grant from the University Düsseldorf (Foko) obtained by Dr. Sydlik and by the German Research Foundation (DFG, GK 1427).
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