Lung mRNA levels of chemokines following HDM sensitization and exposure to MWCNTs.

<p>(A) Lung mRNA levels of CXCL1 at 1 day and 21 days in mice sensitized with HDM allergen with or without MWCNT exposure. (B) Lung mRNA expression of CXCL2. (C) 1 day and 21 day mRNA expression of CCL2 in the lung. Statistical analysis performed using an unpaired student t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparison between treatment groups. ^^^P < 0.001. ^^^^P < 0.0001 for comparison between timepoints. N = 11–14 animals for all 1 day results. N = 8–13 animals for all 21 day results.</p

Lung mRNA levels of chemokines following HDM sensitization and exposure to MWCNTs.

<p>(A) Lung mRNA levels of CXCL1 at 1 day and 21 days in mice sensitized with HDM allergen with or without MWCNT exposure. (B) Lung mRNA expression of CXCL2. (C) 1 day and 21 day mRNA expression of CCL2 in the lung. Statistical analysis performed using an unpaired student t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparison between treatment groups. ^^^P < 0.001. ^^^^P < 0.0001 for comparison between timepoints. N = 11–14 animals for all 1 day results. N = 8–13 animals for all 21 day results.</p

Schematic illustration of a hypothetical mechanism for suppression of MWCNT-induced inflammasome activation and IL-1β secretion during allergic airway inflammation and fibrosis.

<p><i>In vitro</i> experiments using THP-1 monocytic cells differentiated to macrophages showed that the Th2 cytokines IL-4 and IL-13 suppress levels of intracellular pro-caspase-1 and reduce MWCNT-stimulated IL-1β secretion without affecting levels of pro-IL-1β. <i>In vivo</i> experiments with mice pre-sensitized to house dust mite allergen also showed suppression of MWCNT-induced IL-1β and pro-caspase-1 in the lungs, and revealed that MWCNT-induced airway fibrosis was enhanced by allergen challenge. Collectively, <i>in vitro</i> and <i>in vivo</i> experiments suggest that MWCNT-induced inflammasome suppressed in an allergic inflammatory microenvironment could play a role in increased airway fibrogenesis.</p

Inhibition of STAT6 in THP-1 cells increases activity of cleaved, active caspase-1.

<p>Caspase-1 activity assay measuring cleaved, active caspase-1 in cell lysates from THP-1 cells pre-treated with Leflunomide or JAK Inhibitor I, primed with LPS, and exposed to IL-4 and IL-13 and/or MWCNTs. Statistical analysis was performed using a one-way ANOVA with a <i>post hoc</i> Tukey. *P < 0.05, **P < 0.01, ****P < 0.0001 for inhibitor-treated cells compared to control within each treatment group. Data is representative of duplicate experiments.</p

Th2 cytokines suppress pro-caspase-1 without affecting levels of pro-IL-1β.

<p>(A) Taqman quantitative RT-PCR results showing mRNA expression of pro-IL-1β in THP-1s exposed to 100 μg/mL MWCNTs with IL-4, IL-13, or IL-4 and IL-13 co-treatment. B) Taqman qRT-PCR results showing mRNA levels of pro-caspase-1 in THP-1s exposed to MWCNTs with IL-4, IL-13, or IL-4 and IL-13 co-treatment. Statistical analysis was performed using a one-way ANOVA with a <i>post hoc</i> Tukey. ***P < 0.001 for comparison between treatment groups. Values directly above bars indicate significant difference from LPS treatment alone. Values above connecting lines indicate significant difference compared to MWCNT treatment. Data is pooled from three replicate experiments. (C) Representative Western blots showing protein levels of pro-IL-1β, pro-caspase-1, phosphorylated (p)-STAT-6, STAT-6, and β-actin after 24 hour exposure of LPS-primed THP-1 cells to MWCNTs in the absence or presence of IL-4, IL-13, or a combination of IL-4 and IL-13. (D) Quantitative densitometric analysis of pro-IL-1β Western blot signal from two replicate experiments. (E) Quantitative densitometric analysis of pro-caspase-1 Western blot signal from three replicate experiments. Statistical analysis performed using an unpaired student t-test. *P < 0.05 compared to LPS priming alone.</p

Pro-caspase-1 protein levels at 1 day in the lungs of mice are induced by MWCNTs but suppressed by HDM sensitization.

<p>Formalin-fixed, paraffin-embedded lung sections stained with an anti-pro-caspase-1 antibody or a vehicle control. Lung sections from caspase-1 knock-out (KO) mice were used as a negative control. (A) Pro-caspase-1 immuno-staining was observed in the airway epithelium at low magnification (20X). (B) Higher magnification (40X) images showing airway epithelium and alveolar macrophages staining. (C) Quantification of pro-caspase-1 staining in lung sections. Data is pooled from measurements of three individual airways per treatment group. Statistical analysis performed using a one-way ANOVA with a <i>post-hoc</i> Tukey. ***P < 0.001. Values directly above bars indicate a significant difference compared to control animals. Values above connecting lines indicate a significant difference between MWCNT and HDM/MWCNT treatments.</p

Effect of MWCNTs on the lung inflammatory response in mice after sensitization with HDM allergen.

<p>(A) Experimental protocol. (B) Total inflammatory cell numbers in the BALF at 1 day and 21 days in all treatment groups. ****P < 0.0001 for HDM/MWCNT mice compared to control animals. **P < 0.01 for HDM/MWCNT mice at 1 day compared to 21 days. (C) Cytospins of BALF showing neutrophilic inflammation with MWCNTs alone and mixed neutrophilic/eosinophilic inflammation with MWCNTs after HDM sensitization. Images taken at 100X magnification. (D) Relative number of inflammatory cell types counted in BALF cytospins at 1 and 21 days after MWCNT exposure with or without HDM. ***P < 0.001, **P < 0.01, *P < 0.05. Values directly above bars indicate significant differences compared to control animals. Values above connecting lines indicate significant difference between MWCNT, HDM, or HDM/MWCNT treatment groups. Statistical analysis for total cell counts and cell differentials performed using a one-way ANOVA with a <i>post-hoc</i> Tukey. N = 11–14 animals for all 1 day cell differentials and counts. N = 8–13 animals for all 21 day cell differentials and counts.</p

Airway fibrosis at 21 days post-exposure to MWCNTs is increased by pre-sensitization of mice with HDM allergen.

<p>(A) Representative lung sections stained with Masson’s trichrome stain in mice sensitized to HDM and then exposed to MWCNTs. Images taken at 10X magnification. (B) Semi-quantitative morphometric analysis of airway fibrosis. Data is pooled from 4–8 individual airway measurements. Statistical analysis performed using an unpaired student t-test. *P < 0.05 compared to control animals.</p