A Jagged 1-Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles

Abstract

Background: Exposure to traffic-related particulate matter promotes asthma and allergic diseases. However, the precise cellular and molecular mechanisms by which particulate matter exposure acts to mediate these effects remain unclear.

Objective: We sought to elucidate the cellular targets and signaling pathways critical for augmentation of allergic airway inflammation induced by ambient ultrafine particles (UFP).

Methods: We used in vitro cell-culture assays with lung-derived antigen-presenting cells and allergen-specific T cells and in vivo mouse models of allergic airway inflammation with myeloid lineage-specific gene deletions, cellular reconstitution approaches, and antibody inhibition studies.

Results: We identified lung alveolar macrophages (AM) as the key cellular target of UFP in promoting airway inflammation. Aryl hydrocarbon receptor-dependent induction of Jagged 1 (Jag1) expression in AM was necessary and sufficient for augmentation of allergic airway inflammation by UFP. UFP promoted T_H2 and T_H17 cell differentiation of allergen-specific T cells in a Jag1- and Notch 4-dependent manner. Treatment of mice with an anti-Notch 4 antibody abrogated exacerbation of allergic airway inflammation induced by UFP.

Conclusion: UFP exacerbate allergic airway inflammation by promoting a Jag1-Notch 4-dependent interaction between AM and allergen-specific T cells, leading to augmented T_H cell differentiation.

Keywords: Airway hyperresponsiveness; Jagged 1; Notch; Notch 4; allergic airway inflammation; alveolar macrophages; aryl hydrocarbon receptor; asthma; traffic-related particulate matter; ultrafine particles.

Fig. 1

AM differentially uptake nanoparticles and highly express Jag1 in response to UFP. A and B, Flow cytometric analysis of the uptake of fluorescent nanoparticles by different lung cell populations in mice subjected to OVA+UFP-induced allergic airway inflammation. C, Bar graph presentation of the distribution of nanoparticles among lung macrophages (AM and IM), dendritic cells (DC) and neutrophils (Neu). D and E, Relative fold changes in Jag1 transcripts, quantitated by RT-PCR (Fig 1, D), and flow cytometric analysis of Jag1 expression (Fig 1, E), in lung APCs purified from either Il4ra^R576 or Il4ra^R576Lyz2^CreAhr^Δ/Δ mice and treated with PBS or UFP (10 μg/ml). F and G, Relative fold changes in Jag1 transcripts (Fig 1, F), and Jag1 expression (Fig 1, G), in lung APCs purified from either l4ra^R576 or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice and treated with PBS or UFP, as described for Fig 1, D and E. Results are representative of 2 independent experiments. N=3 mice/group. *p<.05; **p<.01; ***p<.001, ***p<.0001 by one-way ANOVA with post-test analysis (panels C, D and F) or Student’s unpaired two-tailed t test (panel F, CD11c⁺ DC group comparison).

Fig. 2

AM support UFP-dependent upregulation of Th cell cytokine production by allergen-specific CD4⁺ T cells in a Jag1-dependent manner. A, Representative flow cytometric analysis of IL-4, IL-13, IL-17 and IFN-γ cytokine production by naive Il4ra^R576CD4⁺DO11.10⁺ T cells co-cultured with FACS-purified AM isolated from l4ra^R576 or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice pulsed with OVA_323-339 peptide in the presence of UFP (10 μg/mL). Cytokine expression was analyzed in gated CD4⁺Foxp3⁻ T cell. B, Frequencies of CD4⁺Foxp3⁻ T cells expressing the respective cytokine upon co-culture with AM that have been either sham treated (PBS) or pulsed with OVA_323-339 peptide, alone or in the presence of UFP. Results are representative of 3 independent experiments. *P < .05, **P < .01, ***P < .001, and ****P < .0001, two-way ANOVA with post-test analysis.

Fig. 3

UFP skews AM-dependent iTreg cell differentiation towards Th2/17 cell like phenotypes in Jag1-dependent manner. Representative flow cytometric analysis and frequencies of CD4⁺Foxp3⁺ iTreg cells (Fig 3, A and B), and their expression of IL-4, IL-13, IL-17, and IFN-g (Fig 3, C-J), on co-culture of naive Il4ra^R576CD4⁺DO11.10⁺ T cells with FACS-purified AM isolated from Il4ra^R576, or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice. The AM that have been either sham treated (PBS) or pulsed with OVA323-339 peptide either alone or in the presence of UFP, as indicated. Results are representative of 3 independent experiments. **P < .01, ***P < .001, and ****P < .0001, two-way ANOVA with post-test analysis.

Fig. 4

Myeloid lineage-specific deletion of Jag1 confers protection against UFP-induced exacerbation of allergic airway inflammation. A, Representative PAS-stained sections of lung isolated from Il4ra^R576 or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice in PBS, OVA or OVA+UFP groups (200X magnification). B, Inflammation scores in the lung tissues isolated from the mouse groups described in Fig 4, A. C–G Airway hyper-responsiveness in response to methacholine (Fig 4, C), absolute numbers of eosinophils (Fig 4, D) and T cells (Fig 4, E) in the BAL fluids, total (Fig 4, F) and OVA-specific (Fig 4, G) levels in the serum of the mouse groups described in Fig 4, A. H–K, Absolute numbers of lung Foxp3⁻CD4⁺T cells secreting IL-4 (Fig 4, H), IL13 (Fig 4, I), IL-17 (Fig 3, J) and IFN-γ (Fig 4, K) in the mouse groups described in Fig 4, A. L–O, Absolute numbers of lung Foxp3⁺CD4⁺Treg cells secreting IL-4 (Fig 4, L), IL13 (Fig 4, M), IL-17 (Fig 4, N) and IFN-γ (Fig 4, O) in the mouse groups described in panel Fig 4, A. Results are representative of 2 independent experiments. N=5 mice/group. *p<0.05, **<0.01, ***<0.001, ****<0.0001 by two-way ANOVA with post test analysis.

Fig. 5

Jag1-sufficient AM rescue UFP-mediated allergic airway inflammation in Il4ra^R576Lyz2^CreJag1^Δ/Δ mice. A, Representative PAS-stained sections of lung tissues of Il4ra^R576Lyz2^CreJag1^Δ/Δ mice supplemented intra-tracheally with AM isolated from Il4ra^R576 or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice that were either sham treated (PBS) or loaded with OVA323-339 peptide (OVA) alone or together with UFP (OVA+UFP). B, Inflammation scores of lung tissues of mice described in Fig 5, A. C–G, Airway hyper-responsiveness (Fig 5, C), numbers of eosinophils (Fig 5, D) and T cells (Fig 5, E) in the BAL fluids, total (Fig 5, F) and OVA-specific (Fig 5, G) levels in the sera of mice described in Fig 5, A. H–K, Numbers of lung Foxp3⁻CD4⁺T cells secreting IL-4 (Fig 5, H), IL13 (Fig 5, I), IL-17 (Fig 5, J) and IFN-γ (Fig 5, K) in the BAL fluids of mice described in Fig 5, A. L–O, Numbers of lung Foxp3⁺CD4⁺Treg cells secreting IL-4 (Fig 5, L), IL13 (Fig 5, M), IL-17 (Fig 5, N) and IFN-γ (Fig 5 O) in the BAL fluids of mice described in Fig 5, A. Results are representative of 3 independent experiments. N=7–12 mice/group. *p<0.05, **<0.01, ***<0.001, ****<0.0001 by two-way ANOVA with Bonferroni post test analysis. n.s.: not significant.

Fig. 6

AM supports UFP-dependent upregulation of Th cell cytokine production by allergen-specific CD4⁺ T cells in a Notch4-dependent manner. A, Representative flow cytometric analysis of IL-4, IL-13 and IL-17 cytokine production by naive Il4ra^R576CD4⁺DO11.10⁺ T cells co-cultured with FACS-purified AM isolated from l4ra^R576 or Il4ra^R576Lyz2^CreJag1^Δ/Δ mice that have been pulsed with OVA_323-339 peptide in the presence of UFP (10 μg/mL). Co-cultures were treated with either isotype control (Iso) Ab or an anti-Notch4 mAb, as indicated, and cytokine analysis was carried out on gated CD4⁺Foxp3⁻ T cell. B, Frequencies of T cells expressing the respective cytokine upon co-culture with AM that have been either sham treated (PBS) or pulsed with OVA_323-339 peptide alone or in combination with UFP (10 μg/mL). Anti-Notch4 mAb or isotype control Ab were added as indicated. Results are representative of 3 independent experiments. *P < .05, **P < .01, ***P < .001, and ****P < .0001, two-way ANOVA with post-test analysis.

Fig. 7

UFP enhances allergic airway inflammation in a Notch4-dependent manner. A, Representative PAS staining of lung tissues isolated from Il4ra^R576 mice sensitized and challenged with OVA alone, or together with UFP, in the presence of either isotype control (Iso) Ab or an anti-Notch4 mAb. B, Inflammation scores in lung tissues of the mouse groups described in in Fig 7, A. C–H Airway hyper-responsiveness in response to methacholine (Fig 7, C), absolute numbers of eosinophils (Fig 7, D), T cells (Fig 7, E) and neutrophils (Fig 7, F) in the BAL fluids, total (Fig 7, G) and OVA-specific (Fig 7, H) levels in the serum of the mouse groups described in Fig 7, A. I-L, Absolute numbers of lung Foxp3⁻CD4⁺T cells secreting IL-4 (Fig 7, I), IL13 (Fig 7, J), IL-17 (Fig 7, K) and IFN-γ (Fig 7, L) in the mouse groups described in Fig 7, A. M–P, Absolute numbers of lung Foxp3⁺CD4⁺Treg cells secreting IL-4 (Fig 7, M), IL13 (Fig 7, N), IL-17 (Fig 7, O) and IFN-γ (Fig 7, P) in the mouse groups described in panel Fig 7, A. Results are representative of 2 independent experiments. N=5 mice/group. *p<0.05, **<0.01, ***<0.001, ****<0.0001 by two-way ANOVA with post test analysis.