Balb/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animal procedures followed the University of California, San Diego, animal-care guidelines. All experiments were performed in at least triplicate.

ISS-ODN (5'-TGACTGTGAACGTTCGAGATGA-3') was purchased from Trilink Biotechnologies (San Diego, CA, USA). OVA was purchased from Sigma-Aldrich (Saint Louis, MS, USA).

Myeloid bone marrow-derived dendritic cells (BMDC) were prepared from Balb/c mice as described previously.7 Briefly, bone marrow from the femurs and tibias of Balb/c mice was plated in petri dishes (Fisher Scientific, Pittsburgh, PA, USA) on day 0 at 2×10⁵ cells/mL in DC medium, which consisted of RPMI (Irvine Scientific, Irvine, CA, USA) supplemented with 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD, USA), 2 mM L-glutamine (Cellgro, Natham, VA, USA), and 100 U/mL penicillin/100 µg/mL streptomycin (Pen/Strep; Cellgro) containing 5 ng/mL recombinant murine GM-CSF (BD PharMingen, San Hose, CA, USA). On day 3, an equal volume of DC medium was added. On day 6, one-half of the volume of DC medium was replaced. Non-adherent cells were harvested on day 7. For plasmacytoid dendritic cells, bone marrow from the femurs and tibias of Balb/c mice was plated in petri dishes (Fisher Scientific, Pittsburgh, PA, USA) on day 0 at 1.5×10⁶ cells/mL in DC medium containing 5 ng/mL recombinant human Flt3 ligand (Peprotech, Rocky Hill, NJ, USA). Non-adherent cells were harvested on day 14. Further purification of pDC cells using anti-CD11c magnetic beads, B220 magnetic beads, and an MACS column (Miltenyi Biotec, Auburn, CA, USA) as per the manufacturer's instructions was performed for selected studies. Flow cytometry, after staining with Abs against the following cell surface markers or appropriate isotype controls (BD PharMingen), was conducted to characterize the cultured cells: CD11c (clone HL3), CD11b (clone M1/70), and B220 (clone RA3-6B2). Single-cell lung suspensions were prepared by collagenase digestion as described previously.8 The DC population was initially enriched by discontinuous density gradient centrifugation using OptiPrep (Axis-Shield PoC AS, Oslo, Norway) according to the manufacturer's instructions. Low-density cells at the interface were harvested, and CD11c+ cells were positively isolated using anti-CD11c magnetic beads (Miltenyi Biotec Inc., Auburn, CA, USA). The CD11c+ population comprised greater than 90% of the total cells as determined by FACS analysis. High-density cells were collected and served as a CD11c-cell population.

Airway inflammation was induced according to a protocol previously described with minor modifications.9 In brief, Balb/c mice were sensitized by subcutaneous injection of 20 µg OVA absorbed with 500 µg alum (Alhydrogel; Brenntag Biosector AS, Federikssund, Denmark) per mouse in saline on days 0, 7, and 14. On days 20 and 25, mice were challenged intratracheally with 10⁶ DC pulsed with or without OVA (Figs. 1A, 2A) or intranasally with 5 µg OVA (Fig. 3A). Mice were then sacrificed on day 26.

To evaluate the role of mDC, mice were divide into groups (n=3) and 10⁵, 3×10⁵, or 10⁶ mDC pulsed with OVA were instilled intratracheally (Fig. 1A). Next, mice were divided into six groups to evaluate the roles of mDC and pDC with or without ISS. mDC alone (group 1), mDC pulsed with OVA (group 2), mDC pulsed with OVA and ISS (group 3), pDC alone (group 4), pDC pulsed with OVA (group 5), or pDC pulsed with OVA and ISS (group 6) were administered intratracheally on days 20 and 25. One million pDC or mDC were administered to each mouse (Fig. 2A). mDC and pDC were pulsed with Ova and/or ISS for 30 min and were washed thoroughly before instillation.

In the final experiment, mice were divided into four groups: group 1 (control, no sensitization), group 2 (sensitization with OVA and alum, PBS challenge), group 3 (sensitization with OVA and alum, OVA challenge, no treatment), and group 4 (sensitization with OVA and alum, OVA challenge, ISS treatment). After sensitization with OVA and alum, ISS-ODN (50 µg/mouse) was administered intraperitoneally 1 day before OVA challenge (day 19).

Twenty-four hours after the last challenge (day 26), mice were subjected to airway responsiveness tests and then sacrificed by CO2 asphyxiation. Five mice were used in each group, and measurements were taken in triplicate.

After sacrifice, bronchoalveolar lavage (BAL) cells and lungs were harvested from each mouse. The total number of cells in BAL was counted, and BAL cells were stained with Wright-Giemsa stain for the differential count. Percentages of eosinophils were determined microscopically using standard morphological criteria.10 The total number of eosinophils was calculated from the total cell number and the percentage of eosinophils in the differential cell count. The levels of each cytokine (IL-4, IL-5, IFN-γ) in BAL fluid were determined by ELISA (BD Biosciences - Pharmingen, San Diego, CA, USA).

Airway responsiveness to methacholine (MCh) was assessed 24 hours after the final OVA challenge using a single-chamber, whole-body plethysmograph obtained from Buxco Electronics Inc. (Wilmington, NC, USA), as described previously.9 The Penh, a dimensionless value, which correlates well with pulmonary resistance measured by conventional two-chamber plethysmography in ventilated mice, was used to monitor airway responsiveness. In the plethysmograph, mice were exposed for 3 minutes to nebulized PBS to establish baseline Penh values and were subsequently exposed to increasing concentrations of nebulized MCh (Sigma-Aldrich) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss Health Care Inc., Somerset, PA, USA). Following each nebulization, recordings were taken for 3 minutes. The Penh values measured during each 3-minute sequence were averaged and are expressed for each MCh concentration as the percentage of baseline Penh values following PBS exposure.

Cells obtained from the lung were stained for CD11c, CD11b, and B220 (BD Biosciences - Pharmingen) and then analyzed using a FACSCalibur flow cytometer (BD Biosciences - Immunocytometry Systems, San Jose, CA, USA).

Data were compared using the Student's t-test, and repeated measures ANOVA was used to compare plethysmography data. A P value of less than or equal to 0.05 was considered statistically significant.

To elucidate the effect of mDC on asthma induction, 10⁵, 3×10⁵, or 10⁶ mDC pulsed with OVA were administered intratracheally (Fig. 1A). mDC induced airway hyperresponsiveness in an mDC number-dependent manner; the difference between groups was statistically significant (P=0.002; Fig. 1B). Eosinophil counts in BAL fluid increased significantly with increasing numbers of mDC (each P<0.05; Fig. 1C). Th2 cytokine (IL-4 and IL-5) levels in BAL also increased significantly and interferon-γ decreased, with increasing mDC administration (Fig. 1D).

To evaluate the effect of pDC on asthma, mDC or pDC (10⁶) with or without OVA were administered intratracheally after sensitization of mice with OVA and alum. To assess the effect of ISS on DC, mDC and pDC were also incubated with ISS prior to intratracheal administration (groups 3 and 6). The experimental protocol is illustrated in Fig. 2A. pDC were isolated using anti-CD11c and anti-B220 MACS beads, and CD11c+ and B220+ groups were isolated with a purity of 94.4% (Fig. 2B). Airway hyperresponsiveness and eosinophil counts induced by mDC (group 2) were abolished with mDC pulsed with ISS administration (group 3; P=0.01 and P<0.001, respectively; Fig. 2C, D). Th2 cytokine levels also significantly decreased in the ISS-pulsed, mDC-administered group (all P<0.01). However, in the pDC-administered groups (groups 4, 5, and 6), airway hyperresponsiveness and eosinophil counts did not differ from those of the non-OVA-pulsed mDC-administered group (group 1), suggesting that priming of T cells by pDC with OVA antigen is poor. IL-4 and IL-5 levels were not significantly different compared to group 1 (all P>0.05).

We also evaluated the in vivo effect of ISS on pulmonary DC populations. ISS was administered to mice on day 19 (Fig. 3A) and the DC population was counted using flow cytometry. The CD11c+CD11b+ cell count increased from 1.84% to 15.80% after OVA challenge and decreased to 9.02% in the ISS-administration group, showing that ISS inhibited migration of mDC populations to the lungs. The proportion of CD11c+B220+ cells increased from 0.60% to 1.08% after OVA challenge, and decreased to 0.69% after ISS administration. In addition, ISS also significantly decreased eosinophil counts and Th2 cytokine (IL-4 and IL-5) levels in the lungs (all P<0.01; Fig. 3C). Interferon-γ levels were not significantly different after ISS administration (P>0.05; Fig. 3C).