Inhaled house dust mite induces pulmonary T helper 2 cytokine production

Abstract

Background: Inhaled house dust mite (HDM) results in T-helper (TH) 2 type pathology in unsensitized mice, in conjunction with airway hyperreactivity and airway remodelling. However, the pulmonary cytokine and chemokine profile has not been reported.

Methods: We have performed a time course analysis of the characteristic molecular mediators and cellular influx in the bronchoalveolar lavage (BAL) and lung in order to define the pulmonary inflammatory response to inhaled HDM extract. Mice were exposed five times a week to soluble HDM extract for 3 weeks. Lung function was measured in groups of mice at intervals following the final HDM challenge. Recruitment of inflammatory cells and inflammatory mediator production was then assessed in BAL and lungs of individual mice.

Results: We found that Th2 cytokines were significantly increased in BAL and lung after HDM challenge from as early as 2 h post-final challenge. The levels of cytokines and chemokines correlated with the influx of eosinophils and Th2 cells to the different compartments of the lung. However, the production of key cytokines such as IL-4, IL-5 and IL-13 preceded the increase in airways resistance.

Conclusion: Inhaled HDM challenge induces a classical Th2 inflammatory mediator profile in the BAL and lung. These data are important for studies determining the efficacy of novel treatment strategies for allergic airways disease.

Fig. 1

House dust mite (HDM)-elicits allergic airway inflammation. Mice were intranasally administered either phosphate-buffered saline (PBS) or 25 μg HDM once daily for 5 days, with 2 days rest, for either 1, 2, 3, 5 or 7 consecutive weeks. Mice were then analysed at these time-points 24 h post-final challenge. (a) Resistance of airways of PBS- and HDM-treated mice. (b) Airway hyperreactivity. RI average was significantly increased 3, 5 and 7 weeks post-challenge compared with PBS, in response to a single dose of methacholine (100 mg/mL). Eosinophils recovered from the (c) lung and (d) bronchoalveolar lavage (BAL) fluid. IL-5 (e and f), IL-13 (g and h) and IL-4 (i and j) measured in the lung (e, g and i) and BALF (f, h and j). Cytokine levels determined by ELISA (n = 18 mice treated with PBS (3–4 mice per group at weeks 1–7). No significant differences were observed between PBS mice at each week therefore data from these control animals were pooled and presented as a single data point for clarity (n = 5–6 mice treated with HDM at each time-point). P<0.05; (Mann–Whitney U-test) compared with PBS. Data are mean±SEM.

Fig. 2

Airway hyperreactivity in house dust mite (HDM)-treated mice. (a) Airway resistance of phosphate-buffered saline (PBS)- and HDM-treated mice at 2, 4, 8 and 24 h post-final challenge. (b) Lung resistance (R_L) was significantly increased 24 h post-challenge compared with PBS, in response to 30 mg/mL methacholine. (c) Airway compliance measured at various time-points post-final challenge. (d) Lung compliance (C_dyn) was significantly decreased at all time-points post-challenge compared with PBS, in response to a single dose of methacholine (30 mg/mL) (n = 12 mice treated with PBS, n = 5–6 mice treated with HDM at each time-point). *P<0.05 (Mann–Whitney U-test) compared with PBS. Data are mean ±SEM.

Fig. 3

Inflammatory cell profile of house dust mite (HDM)-treated mice. Total inflammatory cells, eosinophils, lymphocytes and monocytes and neutrophils recovered from the lung (a, c, e, g) and bronchoalveolar lavage (BAL) (b, d, f, h) 2, 4, 8 and 24 h after the final intranasal challenge (n = 12 mice treated with PBS, n = 5–6 mice treated with HDM at each time-point). *P<0.05; (Mann–Whitney U-test) compared with PBS. †Time-point of peak cellular influx as determined by multiple comparisons between groups. Data averages are median values.

Fig. 4

Inflammatory T cell profile. CD4, CD8 and T1ST2/CD4 positive T cells recovered from the lung (a, c, e) and BAL (b, d, f) 2, 4, 8 and 24 h after the last intranasal challenge [n = 12 mice treated with PBS, n = 5–6 mice treated with house dust mite (HDM) at each time-point]. *P<0.05; (Mann–Whitney U-test) compared with phosphate-buffered saline (PBS). Data averages are median values. †Time-point of peak cellular influx as determined by multiple comparisons between groups.

Fig. 5

Non-classical T cell profile. γδT cells, Th17 cells, CD4, IL-10⁺ T cells and CD25⁺ FoxP3⁺ regulatory T cells recovered from the lung (a, d, g, j) and the bronchoalveolar lavage (BAL) (b, e, h, k) at 2, 4, 8 and 24 h after the final intranasal challenge. A representative FACS plot from the lung cells is shown for each stain (c, CD3+/γδTCR+γδ T cells; f, CD4+/IL-17+T cells; i, CD4+/IL-10+T cells; l, CD25+/FoxP3+Tregs) (n = 12 mice treated with phosphate-buffered saline (PBS), n = 5–6 mice treated with house dust mite (HDM) at each time-point). *P<0.05; (Mann–Whitney U-test) compared with PBS. Data averages are median values. †Time-point of peak cellular influx as determined by multiple comparisons between groups.

Fig. 6

Inflammatory cytokine profile of house dust mite (HDM)-treated mice. IL-4, IL-5, IL-13 and IFN-γ measured in the lung (a, c, e, g) and BAL (b, d, f, h) 2, 4, 8 and 24 h after the final intranasal challenge. No IFN-γ was detected in the bronchoalveolar lavage (BAL) fluid. IL-4, IL-5 and IFN-γ levels were determined by MSD and IL-13 levels by ELISA (n = 12 mice treated with PBS, n = 5–6 mice treated with HDM at each time-point). *P<0.05; (Mann–Whitney U-test) compared with phosphate-buffered saline (PBS). Data are mean ±SEM. †Time-point of peak cellular influx as determined by multiple comparisons between groups.

Fig. 7

Chemokine profile of house dust mite (HDM)-treated mice. Eotaxin-1/CCL11, TARC/CCL17 and KC/CXCL1 levels measured in the lung (a, c, e) and bronchoalveolar lavage (BAL) (b, d, f) 2, 4, 8 and 24 h after the final intranasal challenge. Chemokine levels were determined by ELISA (n = 12 mice treated with PBS, n = 5–6 mice treated with HDM at each time-point). *P<0.05; (Mann–Whitney U-test) compared with phosphate-buffered saline (PBS). Data are mean ±SEM. †Time-point of peak cellular influx as determined by multiple comparisons between groups.

Fig. 8

Eicosanoids are elevated during induction of house dust mite (HDM). (a) 5-HETE and (b) LTB₄ were extracted and quantified 2, 4, 8 and 24 h after the final intranasal challenge. n = 6 *P<0.05; (Mann–Whitney U-test) compared with phosphate-buffered saline (PBS). Data are mean SEM.