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Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

Rydman EM, Ilves M, Koivisto AJ, Kinaret PA, Fortino V, Savinko TS, Lehto MT, Pulkkinen V, Vippola M, Hämeri KJ, Matikainen S, Wolff H, Savolainen KM, Greco D, Alenius H - Part Fibre Toxicol (2014)

Bottom Line: However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

View Article: PubMed Central - PubMed

Affiliation: Nanosafety Research Centre, Finnish Institute of Occupational Health, Helsinki, Finland. elina.rydman@ttl.fi.

ABSTRACT

Background: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.

Methods: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation.

Results: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.

Conclusions: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

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Related in: MedlinePlus

Transcriptome analysis of lung tissue reveals rapid activation of innate immune system in response to inhaled rCNT. a: heatmap of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lungs of mice exposed to rCNT or tCNT for 4 h and sacrificed either immediately after exposure or on the following day. Genes are arranged by hierarchical clustering analysis. Untreated (UT) and tCNT-exposed lung samples share similar expression patterns shortly after 4-h exposure. Red color indicates higher expression while green denotes lower expression. b: Venn diagram of the microarray results summarizing the numbers of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lung tissue after 4 h exposure to CNT. The diagram shows that rCNT cause a markable expression of multiple distinct genes already after 4-h exposure (776 genes) while the number of genes specific to tCNT is notably lower (109 genes). c: pathway bubble-plot shows that inhalation of rCNT activates several innate immunity-associated pathways after the 4 h exposure. The diameter of each circle represents the number of genes annotated in each pathway (legend “count”). Red color indicates up-regulated pathways and green color down-regulated pathways compared to untreated controls.
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Fig4: Transcriptome analysis of lung tissue reveals rapid activation of innate immune system in response to inhaled rCNT. a: heatmap of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lungs of mice exposed to rCNT or tCNT for 4 h and sacrificed either immediately after exposure or on the following day. Genes are arranged by hierarchical clustering analysis. Untreated (UT) and tCNT-exposed lung samples share similar expression patterns shortly after 4-h exposure. Red color indicates higher expression while green denotes lower expression. b: Venn diagram of the microarray results summarizing the numbers of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lung tissue after 4 h exposure to CNT. The diagram shows that rCNT cause a markable expression of multiple distinct genes already after 4-h exposure (776 genes) while the number of genes specific to tCNT is notably lower (109 genes). c: pathway bubble-plot shows that inhalation of rCNT activates several innate immunity-associated pathways after the 4 h exposure. The diameter of each circle represents the number of genes annotated in each pathway (legend “count”). Red color indicates up-regulated pathways and green color down-regulated pathways compared to untreated controls.

Mentions: We further investigated the early regulatory events at the transcriptomic level (NCBI GEO Accession Number GSE50176, Additional file 3). C57BL/6 mice were hence exposed by inhalation to rCNT or tCNT once for 4 h, and sacrificed either immediately or 24 h after the cessation of the exposure. At 4 h, the tCNT treatment had a relatively mild effect on the gene expression and these samples tended to cluster together with the untreated ones (Figure 4a). On the contrary, the effect of rCNT was remarkable at the same time point, impacting many more genes and with much greater amplitude of variation from the baseline samples (Figure 4a,b). At 4 h, rCNT exposure induced almost exclusively pathways essential for the innate immunity (e.g. NOD-like receptor and Toll-like receptor signaling pathways, Chemokine signaling pathways and Cytokine-cytokine receptor interaction pathways) (Figure 4c). Moreover, cancer related pathways as well as insulin signaling pathways were up-regulated after rCNT exposure. In sharp contrast, the innate immunity pathways were either not activated or were down-regulated after a 4 h inhalation of tCNT (Figure 4c). At 24 h following the 4 h exposure, a stronger effect of tCNT took place on a wider set of genes, causing changes similar in size and range of variation to those elicited by rCNT. Nonetheless, the gene expression signatures of rCNT and tCNT were quite distinct, as the group of the genes with similar behavior in both treatments was relatively small (Figure 4b, Additional file 4). In addition, direct comparison of the transcriptome of rCNT and tCNT exposed mice at each time point revealed that innate immunity pathways and chemokine-cytokine signaling pathways were significantly more expressed after rCNT, whereas lysosomal activity and carbohydrate metabolism pathways were induced by tCNT (Additional file 5). Some highly significant genes in the microarray analysis also relevant to asthma (Ccl2, Ccl11, Ccl24 and Cxcl5) were further confirmed by quantitative PCR (Additional file 6).Figure 4


Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

Rydman EM, Ilves M, Koivisto AJ, Kinaret PA, Fortino V, Savinko TS, Lehto MT, Pulkkinen V, Vippola M, Hämeri KJ, Matikainen S, Wolff H, Savolainen KM, Greco D, Alenius H - Part Fibre Toxicol (2014)

Transcriptome analysis of lung tissue reveals rapid activation of innate immune system in response to inhaled rCNT. a: heatmap of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lungs of mice exposed to rCNT or tCNT for 4 h and sacrificed either immediately after exposure or on the following day. Genes are arranged by hierarchical clustering analysis. Untreated (UT) and tCNT-exposed lung samples share similar expression patterns shortly after 4-h exposure. Red color indicates higher expression while green denotes lower expression. b: Venn diagram of the microarray results summarizing the numbers of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lung tissue after 4 h exposure to CNT. The diagram shows that rCNT cause a markable expression of multiple distinct genes already after 4-h exposure (776 genes) while the number of genes specific to tCNT is notably lower (109 genes). c: pathway bubble-plot shows that inhalation of rCNT activates several innate immunity-associated pathways after the 4 h exposure. The diameter of each circle represents the number of genes annotated in each pathway (legend “count”). Red color indicates up-regulated pathways and green color down-regulated pathways compared to untreated controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Transcriptome analysis of lung tissue reveals rapid activation of innate immune system in response to inhaled rCNT. a: heatmap of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lungs of mice exposed to rCNT or tCNT for 4 h and sacrificed either immediately after exposure or on the following day. Genes are arranged by hierarchical clustering analysis. Untreated (UT) and tCNT-exposed lung samples share similar expression patterns shortly after 4-h exposure. Red color indicates higher expression while green denotes lower expression. b: Venn diagram of the microarray results summarizing the numbers of differentially expressed genes (linear FC > /1.5/, post hoc adjusted P-value = 0.01) in lung tissue after 4 h exposure to CNT. The diagram shows that rCNT cause a markable expression of multiple distinct genes already after 4-h exposure (776 genes) while the number of genes specific to tCNT is notably lower (109 genes). c: pathway bubble-plot shows that inhalation of rCNT activates several innate immunity-associated pathways after the 4 h exposure. The diameter of each circle represents the number of genes annotated in each pathway (legend “count”). Red color indicates up-regulated pathways and green color down-regulated pathways compared to untreated controls.
Mentions: We further investigated the early regulatory events at the transcriptomic level (NCBI GEO Accession Number GSE50176, Additional file 3). C57BL/6 mice were hence exposed by inhalation to rCNT or tCNT once for 4 h, and sacrificed either immediately or 24 h after the cessation of the exposure. At 4 h, the tCNT treatment had a relatively mild effect on the gene expression and these samples tended to cluster together with the untreated ones (Figure 4a). On the contrary, the effect of rCNT was remarkable at the same time point, impacting many more genes and with much greater amplitude of variation from the baseline samples (Figure 4a,b). At 4 h, rCNT exposure induced almost exclusively pathways essential for the innate immunity (e.g. NOD-like receptor and Toll-like receptor signaling pathways, Chemokine signaling pathways and Cytokine-cytokine receptor interaction pathways) (Figure 4c). Moreover, cancer related pathways as well as insulin signaling pathways were up-regulated after rCNT exposure. In sharp contrast, the innate immunity pathways were either not activated or were down-regulated after a 4 h inhalation of tCNT (Figure 4c). At 24 h following the 4 h exposure, a stronger effect of tCNT took place on a wider set of genes, causing changes similar in size and range of variation to those elicited by rCNT. Nonetheless, the gene expression signatures of rCNT and tCNT were quite distinct, as the group of the genes with similar behavior in both treatments was relatively small (Figure 4b, Additional file 4). In addition, direct comparison of the transcriptome of rCNT and tCNT exposed mice at each time point revealed that innate immunity pathways and chemokine-cytokine signaling pathways were significantly more expressed after rCNT, whereas lysosomal activity and carbohydrate metabolism pathways were induced by tCNT (Additional file 5). Some highly significant genes in the microarray analysis also relevant to asthma (Ccl2, Ccl11, Ccl24 and Cxcl5) were further confirmed by quantitative PCR (Additional file 6).Figure 4

Bottom Line: However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

View Article: PubMed Central - PubMed

Affiliation: Nanosafety Research Centre, Finnish Institute of Occupational Health, Helsinki, Finland. elina.rydman@ttl.fi.

ABSTRACT

Background: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.

Methods: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation.

Results: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.

Conclusions: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Show MeSH
Related in: MedlinePlus