Preventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

Ectoine application reduces neutrophilic lung inflammation induced by CNP in Balb/c mice. a Experimental design, animals (n = 5) were exposed to PBS (control), 2.5 mg/kg CNP, or 2.5 mg/kg CNP with 1 mM ectoine (E) and subsequently sacrificed at the indicate time points. b Differential cell numbers in BAL (means, SEM). c CXCL1 levels in BAL. * significant differences were observed in total cell numbers, neutrophil numbers and CXCL1 levels (p < 0.05, Mann Whitney U-test)

Fig. 2

CNP exposure during sensitization leads to enhanced immune responses which can be attenuated by ectoine. a Experimental design, for sensitization, animals (n = 8) were treated with PBS, CNP, and CNP + E as described in Fig. 1. Additionally, 12 h after this treatment animals received 20 μg OVA. At day 21 serum was collected (S). After challenge (1 % OVA in PBS, 30 min) on three consecutive days (d32 – d34), animals were sacrificed and BAL, blood and lymph nodes were collected. b OVA-specific IgE prior to and after challenge. c Differential cell counts (representative plots and means, SEM) after challenge significance bar indicates differences in total cell numbers and neutrophil numbers. d CXCL1 levels in BAL after challenge. *p < 0.05, Mann Whitney U-test

Fig. 3

Changes in lymph node responses after challenge. Lymph nodes from the animals (Fig. 2) were analysed for adaptive immune responses. a Total cell numbers in peribronchial lymph nodes (representative plots and means, SEM). b IL-4 release in re-stimulated lymph node cells. c IL-13 release in re-stimulated lymph node cells. *p < 0.05, Mann Whitney U-test

Fig. 4

Ectoine application reduces the frequency of antigen loaded dendritic cells in lymph nodes. a Animals (n = 3 in control groups, n = 8 in exposure groups) were exposed to 50 μg Alexa Fluor 488-labelled OVA 12 h after the application of PBS (control), CNP, or CNP + E. In order to control for effects of labelled OVA, an additional PBS group without OVA was employed. b BAL cell numbers (means, SEM) 36 h after initial treatment. c Total lymph node cells. d Percentage of OVA-488 positive dendritic cells (MHCII⁺, CD11c⁺). e. Percentage of OVA-488 positive macrophages (MHCII⁺, F4/80⁺). *significant differences were observed in total cell numbers, neutrophil numbers, and percentage of OVA-488 positive dendritic cells (p < 0.05, Mann Whitney U-test)

Fig. 5

Effects of CNP and lavage fluid on bone marrow derived dendritic cells. Dendritic cells derived from Balb/c mice (n = 7) were exposed to the indicated doses of CNP (a) or lavage fluid (b) from exposed animals. c Representative histograms determining CCR7⁺ cells. *significantly different from PBS alone, # significantly different from cells treated with BAL from CNP-exposed animals. (p < 0.05, ANOVA with post hoc testing)