Hopefully, future studies will help to clarify the potential usefulness of chitin as active component for novel immunosuppressive therapeutic strategies. IL-4 reporter mice (4get mice) were kindly provided by R. M. Locksley (UC San Francisco) 38. These mice carry an IRES-eGFP construct inserted after the stop codon of the IL-4 gene. B7-H1−/− mice were kindly provided by L. Cheng (Johns Hopkins University) 34. TLR2−/−39 and TLR4−/−

mice were obtained from C. Kirschning (TU München). MyD88−/− and MyD88/TRIF−/− mice were obtained from H. Wagner (TU München). TLR3−/− mice were obtained from S. Akira. Stat6−/− mice 40, DO11.10 TCR-tg mice 41 and BALB/c mice were originally obtained from The Jackson Laboratory (Bar Harbour, ME). Single-cell suspensions of spleen and mesenteric LN from Dactolisib mw DO11.10/4get mice were prepared and 1×106 TCR-tg cells were transferred into BALB/c recipient mice. One and two days later, mice received intranasal applications of 500 μg OVA (Sigma-Aldrich,

St. Louis, MO) in 50 μL PBS with or without chitin powder (10 mg/mouse). Mice were analyzed on day 5 after T-cell transfer by flow cytometry. Purified chitin from crab shells was used (C9752, Sigma-Aldrich). The colloidal chitin powder is chemically identical to native chitin and was generated by methanesulfonic acid treatment as described previously 42. In total, 10 mg chitin powder or glass beads (10–50 μm; Kisker, Germany) were suspended in 500 μL PBS and left at room temperature for 2 min to allow sedimentation of large particles. The supernatants were collected and washed once with PBS by centrifugation at 14 000 rpm followed by resuspension of the pellet in 500 μL PBS. The suspensions were CP-868596 clinical trial stored at 4°C until setup of the experiments. The E-toxate test (Sigma-Aldrich) was used to exclude contamination with LPS. Macrophages were differentiated from BM cells in RPMI 1640 (PanBiotech, Aidenbach, Germany) Tau-protein kinase supplemented with 10% FCS (Invitrogen, Carlsbad, CA), 2 mM L-glutamine, 100 U/mL penicillin,

100 μg/mL streptomycin (Biochrom AG, Berlin, Germany) and 5×10−5 M β-mercaptoethanol (Merck, Darmstadt, Germany) for 8 days in the presence of 10% supernatant from the M-CSF producing fibroblast cell line L929. Macrophages were scraped off the plates and cultured for 24 h in the presence of chitin- or glass-suspensions which covered about 50% of the surface of the culture plate. Untouched polyclonal CD4+ T cells were isolated by MACS technology (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) from 4get mice and cultured in 170 μL RPMI 1640 and 10% FCS under neutral (20 ng/mL IL-2) or Th2-polarizing conditions (20 ng/mL IL-2, 10 ng/mL IL-4 and 10 μg/mL anti-IFN-γ (clone XMG1.2)) at 2×106 cells per well in a flat-bottom 96-well plate which had been coated for 24 h at 4°C with anti-TCR (1 μg/mL) and anti-CD28 (1 μg/mL) mAb. Briefly, 30 μL resuspended chitin or glass beads or PBS were added to the cultures which were then analyzed on day 4 by flow cytometry.