Fig 1.
IL-33 was significantly increased in lungs of HDM mouse model.
Concentrations of (a) IL-33, (b) IL-25 in the lung tissue were measured by ELISA (n = 6 in each group). (c) Thymic stromal lymphopoietin (TSLP) level in the lung tissue was measured by real-time RT-PCR assay (n = 6 in each group), because TSLP protein concentration was below the detection level. (d) Immunohistochemistry (IHC) examination of anti-normal goat IgG control and anti-IL-33 antibody in the lung section. Original magnifications were × 200 and × 500. (e) The number of IL-33 positive cells by IHC (6 sections were counted by two pulmonologists).
Fig 2.
Flow cytometry analysis of Ly6c-positive monocytes in lungs of HDM mice and control mice.
(a) Cells were identified from digested lungs, and after exclusion of doublets and debris, leukocytes were separated by CD45 staining. CD11c-negative cells were identified to investigate Ly6c-positive monocytes. (b) Flow cytometry analysis of Ly6c-positive monocytes (CD45+, CD11c-, CD11b+, Ly6c+) in lungs of HDM mice and control mice. (c) The percentage of Ly6c-positive monocytes in lung were compared in HDM mice and control mice (n = 6 in each group). **P<0.01
Fig 3.
Lung mononuclear cells which positively expressed Ly6c are a source of IL-33.
Lung mononuclear cells were collected from lung tissue as described in the text. Briefly, isolated single cells from lung tissue were resuspended at 2.0 × 106 cells/ml in RPMI1640 medium supplemented with 10% FCS for 24 hours. Adherent cells were harvested and used as mononuclear cells in the experiments. (a) Immunofluorescence examination of lung mononuclear cells in HDM mice. Cells were stained with anti-F4/80 antibody (right upper panels) and anti-IL-33 antibody (left lower panels) and images were merged (right lower panels). Original magnification was × 800. (b) Lung mononuclear cells were lysed by freezing and thawing to extract protein. Concentrations of IL-33 in the lung mononuclear cells were measured by ELISA (n = 6 in each group). (c) Lung mononuclear cells were stained for Ly6c and analyzed by flow cytometery. Representative histograms of Ly6c+ cells in control mice and HDM mice are shown. (d) The percentage of Ly6c+ cells were counted by flow cytometery. **P<0.01.
Fig 4.
The protocols for HDM-induced airway inflammation and clodronate treatment of mice.
Mice were sensitized intranasally with 25 μg HDM or vehicle on days 1, 8, and 15. Mice were challenged intranasally with 5 μg HDM on days 22, 23, and 24. Four hours later after final challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were harvested for further analysis. Clophosome-A, liposomal clodronate, and plain control liposomes for clophosome (150μl / animal) were administered by intravenous injection on days 0, 7, 14, 21, 22, and 23. On the day when HDM was given, clodronate and control liposomes were administered 30 minutes before the HDM inoculation.
Fig 5.
Clodronate-liposome attenuated the HDM induced airway inflammation, airway hyper reactivity and production of Th2 cytokines.
(a) Total and differential cell counts in bronchoalveolar lavage fluids (n = 6 in each group). (b) Airway hyper reactivity was measured by assaying airway resistance during graded concentration of methacholine (n = 6 in each group). #p<0.05 HDM+emp. lipo group compared with PBS+emp. lipo group. ##p<0.05 HDM+CL. lipo group compared with HDM+emp. lipo group. (c) Histological examination of airway inflammation. Sections were stained with H&E (upper panels) or PAS (lower panels). Original magnification was × 200. Empty liposome control (emp. lipo) and clodronate liposome (CL) results are shown. Concentrations of (d) IL-4, (e) IL-5, and (f) IL-13 in the lung tissue were measured by ELISA (n = 6 in each group). **P<0.01, *P<0.05
Fig 6.
Lung mononuclear cells expressed M2 markers but not M1 markers.
The expression of TNF (a), IL-1β (b), arginase-1 (c), and YM-1 (d) levels in the lung mononuclear cells was measured by real-time RT-PCR assay.
Fig 7.
Clodronate attenuated the IL-33 in lungs of HDM mice.
(a) Concentrations of IL-33 in the lung tissue were measured by ELISA in 3 groups (intranasal PBS and intravenous empty liposomes, HDM and empty liposomes, HDM and clodronate liposomes: n = 6 in each group). (b) Immunohistochemistry (IHC) examination of IL-33 in the lung sections. Original magnification was × 200. (c) IL-33-positive cell counts by IHC (6 sections were counted by two pulmonologists). **P<0.01.