Nasal immunization with rLdpA-chitin complex induced a Th2 immune response.

(A) rLdpA (10 μg), chitin (100 μg), rLdpA–chitin complex (rLdpA, 10 μg; chitin, 100 μg), CpG-ODN (20 μg), rLdpA+CpG-ODN (rLdpA, 10 μg; CpG-ODN, 20 μg), or vehicle (PBS) was administered intranasally to BALB/c mice three times. All animals were sacrificed at 72 h after the last nasal administration, and samples were obtained. (B) The numbers of total cells, neutrophils (CD11c− Siglec F− CD11b+ Ly-6G+), and eosinophils (CD11c− Siglec F+) in bronchoalveolar lavage (BAL) fluid were measured by flow cytometry. (C) mRNA expression levels of Kc/Cxcl1 and Ccl11/Eotaxin-1 in the lung were measured by quantitative real-time PCR (qRT-PCR). The mRNA levels of these genes were normalized relative to Gapdh mRNA. The fold differences in expression are shown relative to the vehicle control group. (D) IL-4, IL-5, and IFN-γ secretion by mediastinal lymph node (MLN) cells after ex vivo re-stimulation with or without 10 μg/mL rLdpA. (E) Histological examination of lung tissues stained with Periodic acid–Schiff (PAS). Scale bar, 100 μm. (F) mRNA expression levels of Muc5b and AMCase in the lung were measured by qRT-PCR. The mRNA levels of these genes were normalized relative to Gapdh mRNA. The fold differences in expression are shown relative to the vehicle control group. (G) Levels of serum LdpA-specific IgG1, IgG2a, and IgE measured by indirect ELISA. Data are shown as the mean ± standard deviation (SD) (n = 6–7 mice/group). Each symbol represents an individual sample.