asahii. Recently, it has been shown that MyD88-deficient mice develop severe intestinal
inflammation, indicating that MyD88 signaling plays an important protective role. This raises the possibility that Gal-9 up-regulates the immunosuppressive CD11b+Ly-6Chigh Mϕ or pDC-like Mϕ differently depending on the pathogenic circumstances (T. asahii versus tumor), because T. asahii appears to activate MyD88 through Dasatinib TLR on those cells. Collectively, the studies presented here indicate that infiltration of CD11b+Ly-6Chigh Mϕ, probably MDSC, into the lung at the early phase of experimental HP suppresses the severity of experimental HP. In addition, Gal-9 expands CD11b+Ly-6Chigh Mϕ with suppressive activity on Th cell functions in BM cells. Female C57BL/6 mice (7–8 weeks old) were obtained from Charles River Laboratories Japan (Yokohama, Japan). Animals were kept in accordance with international guidelines and national law. The protocol of this study was approved by the Kagawa University Animal Care and Use Committee. Expression and purification of recombinant human stable Gal-9 was described previously 32, 33. All Gal-9 preparations used in this report were >95% pure by SDS-PAGE with less than 0.3 endotoxin units/mL (<0.03 ng/mL),
as assessed by a limulus turbimetric kinetic assay using a Toxinometer ET-2000 (Wako, Osaka, Japan). Protein concentration was determined with a bicinchoninic acid assay reagent (Pierce, Rockford, IL, USA), using BSA as a standard. Particulate
GDC-0449 in vivo T. asahii, an etiologic agent of HP, was prepared as previously described 34. The powdered material was suspended in sterile PBS (pH 7.4) at a concentration of 4 mg/mL and stored at −20°C until use. Mice were intranasally sensitized with 50 μL (200 μg/mouse) of T. asahii Ag three times daily. After 14 days, mice were challenged once with 50 μL (200 μg/mouse) of the Ag. Mice were simultaneously given either recombinant Gal-9 (0.3, 3, and 30 μg/mouse) or PBS subcutaneously. Differential cell counts for each mouse used Diff Quik staining mafosfamide (Baxter, McGaw Park, IL, USA) or Giemsa staining. Sections of left lungs were stained with hematoxylin and eosin. Histological scores were graded from 0 to 4 as described previously 35; 0: no inflammatory cells, 1: <10%, 2: 10–25%, 3: 25–50%, and 4: >50%. IL-2, TNF-α, IL-12p40, IFN-γ, IL-17, IL-1β, IL-4, IL-6, and IL-13 contents in BALF and culture supernatants were assayed by quantitative ELISA for murine cytokines/chemokines using cytokine-specific kits (R&D Systems, Minneapolis, MN, USA) as described previously 7. BALF cells obtained from mice were washed in PBS with 0.5% FBS and incubated with appropriate fluorochrome-labeled antibodies, then analyzed by flow cytometry using a Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, CA, USA).