SpA/CFA (sc) SpA/IFA (sc) SpA (in) X Days 0 14 28 30 27 29 31 32 Neutralizing Abs (ip or in) Supplementary Fig. 1. Experimental protocol for SpA-induced asthma in mice Mice immunized subcutaneously (sc) with SpA in CFA followed by a boost at day 14 were administered SpA intranasally (in) for three consecutive days (days 28, 29 and 30). In some experiments SpA-immunized and -boosted mice were administered neutralizing anti-IL-18 ip, or soluble IL-13R2 or anti-IFN- intranasally (in) at days 27 and 29.
B C A PBS OVA SpA * * * * * * * * * Supplementary Fig. 2. OVA challenge fails to induce asthmatic inflammation in SpA-immunized mice. Mice immunized subcutaneously (sc) with SpA in CFA followed by a boost at day 14 were administered PBS (A), OVA (B) or SpA (C) intranasally for three consecutive days. SpA challenge caused eosinophilic accumulation in BALF (C). In contrast, PBS or OVA challenge could not at all (A, B). Asterisks indicates eosinophils.
15 SpA SpA+IL-13R2 10 SpA+IFN- change of sRAW PBS 5 0 0 5 10 20 -methacholine (mg/ml) Supplementary Fig. 2 Supplementary Fig. 3 Importance of IL-13 but not IFN- for SpA-induced AHR SpA-immunized mice were additionally administered without (SpA) (squares in red) with soluble decoy IL-13R2 (circles in green) or neutralizing anti-IFN- (IFN-) (diamonds in orange). After intranasal challenge with SpA or PBS (triangles in blue), mice were analyzed for AHR with non-invasive method as shown previously (Hayashi N, et al. J. Exp. Med. (2004) 199:535). Blockade of IFN-, but not IL-13, could prevent the AHR.
30 25 20 15 IFN-(ng/ml) 10 5 0 CD3 hIL-18 hIL-18 Supplementary Fig. 4. Neutralizing activity of anti-human IL-18 mAb CD4+ T cells were isolated from PBMCs of volunteers and incubated under Th1 conditions for two rounds. Human Th1 cells were incubated with immobilized anti-CD3 and human rIL-18 (500 ng/ml) in the absence or presence of anti-human IL-18 mAb (30 g/ml) for twenty four hours. Human IFN- concentrations were measured by ELISA.
Supplementary Fig. 5. Human PBMC produce IL-13 and IFN- in response to SpA PBMC (1 × 106/ml) were incubated with 100 g/ml of SpA for four days. Human IL-18, IL-12, IL-13, IL-4 and IFN- concentrations in each supernatant were measured by ELISA. Similar results were obtained in three separate experiments. ND = not detected.
C-/-Rag2-/-mice transferred with PBS SpA-stimulated hPBMC Lung Liver (x100) Supplementary Fig. 6. Abnormal cell infiltration in lung and liver of transiently humanized mice PBMC (1 × 106/ml) were incubated with 100 g/ml of SpA for four days. SpA-stimulated PBMC or PBC were transferred into C-/-Rag2-/- mice, and lung and liver specimens were sampled at day 7 for histological study. Many infiltrates were found in those organs of the transiently humanized mice.