House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells

Abstract

Barrier epithelial cells and airway dendritic cells (DCs) make up the first line of defense against inhaled substances such as house dust mite (HDM) allergen and endotoxin (lipopolysaccharide, LPS). We hypothesized that these cells need to communicate with each other to cause allergic disease. We show in irradiated chimeric mice that Toll-like receptor 4 (TLR4) expression on radioresistant lung structural cells, but not on DCs, is necessary and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony-stimulating factor, interleukin-25 and interleukin-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM-driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma, including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.

Figure 1. Assesment of the reconstitution rate of chimeric animals

(a) Various chimeric mice (coded as bone marrow donor genotype ->recipient genotype) were prepared. (b) The TLR4 expression in the airways of the diverse chimeric mice was assessed using immunostaining and studied by confocal imaging (purple). This image was merged with a bright field image. (c) The degree of chimerism was analyzed by determining the percentage of GFP⁺ B cells and DC in lymph node and alveolar macrophages of WT^MHCIIgfp->WT mice (gray histogram). The background for GFP positivity was set on fluorescence intensity of mice receiving GFP-negative bone marrow; black histogram). These experiments were done 3 times.

Figure 2. TLR4 expression on radioresistant stromal cells is necessary and sufficient for recruitment of DCs to the lungs in response to LPS

Various chimeric mice (coded as donor genotype ->recipient genotype) were injected i.t with LPS or PBS. The recruitment of (a) BAL fluid neutrophils, (b) BAL fluid monocytes, (e) tracheal digest MHCII+CD11c+ dendritic cells and (f) MHCII+CD11c+CD11b+ DCs was analyzed 24h later. The tracheal counts have been corrected for 1.10⁶ CD45^neg structural cells to correct for differently sized explants. Production of KC chemokine for neutrophils (c) and CCL2 for monocytes and DCs (d) was measured in BAL fluids. *:p<0.05. This is a representative experiment out of 4. Four-six mice/group were used

Figure 3. TLR4 expression on radioresistant stromal cells is necessary and sufficient for activation of mucosal DCs

(a)WT->WT and WT->TLR4^−/− chimeras were injected i.t with LPS or PBS. Tracheal explants were prepared 2hours later and analyzed by two-photon microscopy. Green DCs are located within red epithelial cells. Nuclei were counterstained in blue. (b) Levels of GM-CSF measured 24 h after LPS exposure. (c) Chimeric mice were injected i.t with fluorescent OVA^AF647 together with LPS or PBS. The number of OVA⁺ migrating DCs was enumerated in the MLNs thirty-six hours later. (d) On day 0, WT->WT and WT->TLR4^−/−- chimeras received an intravenous injection of CFSE-labelled OVA-specific naive OTII T cells, and were administered with OVA/LPS or OVA/PBS i.t on day 1. T cell proliferation was evaluated in the MLNs at day 5. Proliferation index for each group is shown in the upper right corner of the histograms. (e) Cytokine production by MLN cells 5 days after OVA/LPS administration. *:p<0.05. These panels show one representative out of 2–4 experiments. 4–6 mice/group were used.

Figure 4. TLR4 expression on airway structural cells is necessary and sufficient for an innate immune response to HDM allergen

Chimeras were injected i.t with PBS or HDM allergen. The number of MHCII+CD11c+ (a) MHCII+CD11c+CD11b+ (b) DCs in tracheal digests was analyzed 24 hours later. The tracheal counts have been corrected for 1.10⁶ CD45^neg structural cells to correct for differently sized explants. (c) Production of GM-CSF, (D) TSLP, (E)IL-25 and (F) IL-33 was measured in BAL fluids. *:p<0.05. This is one representative experiment out of 3. 5–6 mice/group were used

Figure 5. TLR4 expression on airway structural cells is necessary and sufficient for house dust mite driven Th2 responses and allergic inflammation

Chimeras were injected i.t with HDM allergen on days 0, 7, and 14. (a) BAL fluid was analyzed by flow cytometry 72h after challenge. (b) Cytokine measurements of BAL fluids. (c) Cytokine measurements of MLN cells restimulated in vitro for 5 days with 30mg/ml HDM. (d) Hematoxylin/eosin staining of lung sections. These experiments were performed 2 times with 4–6 mice/group. One representative experiment is shown

Figure 6. Intrapulmonary delivery of a TLR4 antagonist reduces HDM driven inflammation and airway hyperresponisveness

C57Bl/6 mice were exposed to HDM or to PBS (Control) on day 0. All the mice were then challenged with HDM admixed to a TLR4 antagonist or the vehicle on days 7 and 14. (a) Differential cell count of BAL fluid was determined by flow cytometry 72 h after challenge. (b) Cytokine measurements of BAL fluids (c) Hematoxylin staining of lung sections. (d) Lung function was determined by invasive measurement of airway resistance in response to increasing concentrations of metacholine. *, P < 0.05. These experiments were performed 2 times with 6 mice/group. One representative experiment is shown.