Fig 2: OSM, Areg and IL33 are elevated in plasma during Alt/PNE induced food allergy. Plasma was collected at 4 h after oral PNE gavage (treated as in Figure 3D). Plasma (A) OSM, (B) Areg, and (C) IL33 were measured by ELISA. N = 8–10/group. (A,C) *p < 0.05 as compared to the PNE only skin-treated group. (B) *p < 0.05 as compared to the anti-OSM/Alt/PNE treated group. Alt, Alternaria alternata extract; Areg, amphiregulin; IL33, interleukin 33; OSM, oncostatin M; PNE, peanut extract.

Fig 3: S. aureus stimulates RANKL in isolated mouse calvarial osteoblasts independent on cytokine but dependent on prostaglandin productions and TLR2.(A) S. aureus time-dependently stimulated Tnfsf11 mRNA without affecting Tnfrsf11b mRNA in mouse osteoblasts. (B) Concentration-dependent stimulation of Tnfsf11 mRNA, with no effect on Tnfrsf11b mRNA, by S. aureus (3x106 CFU/ml). (C) S. aureus (3x106 CFU/ml) upregulated the mRNA expression of Il1b, Il11, Il6, Lif, Osm and Tnfsf2 in osteoblasts. (D) The stimulatory effect by S. aureus (3x106 CFU/ml) on Tnfsf11 mRNA expression in osteoblasts was unaffected by adding a mixture of antibodies neutralizing IL-1ß, IL-6, IL-11, LIF, OSM and TNF-a. (E) S. aureus (3x106 CFU/ml) stimulated Ptgs2 mRNA in mouse osteoblasts. (F) Indomethacin (1 µmol/l) abolished Tnfsf11 mRNA in osteoblasts induced by S. aureus (3x106 CFU/ml). (G, H) The stimulatory effect by S. aureus (3x106 CFU/ml) on Tnfsf11 and Ptgs2 mRNA was observed in osteoblasts from wild type mice but not from Tlr2 deficient mice. Data are means of 5 observations and SEM is given as vertical bars when larger than the radius of the symbol. In Fig 4A, effects on Tnfsf11 mRNA were statistically significant at 1–48 h (P<0.001). In Fig 4B, effects on Tnfsf11 mRNA were statistically significant at 106 and 3x106 (P<0.01) and at 107 and 3x107 (P<0.001) CFU/ml. ***P<0.001 compared to unstimulated control (D-G) or to S. aureus stimulated osteoblasts (G).

Fig 4: Allergen skin sensitization of FT+/- neonates induced skin expression of Il33, Areg, and Osm. (A) Timeline for mating and for FT+/- pup treatments with saline, Alt, PNE, and Alt/PNE allergens. Tissues were collected 4 h after the last pup skin sensitization. Relative expression of skin expression of (B) Osm, (C) Areg, and (D) Il33 as determined by RT-qPCR. N = 8–10/group. The data in all figures include both genders for pups because there were no differences in outcomes by gender as we previously reported for Alt/PNE skin sensitization (19). *p < 0.05 as compared to the saline skin-sensitized groups or WT groups. Alt, Alternaria alternata extract; Areg, amphiregulin; FT-/-, flaky tail mice homozygous for filaggrin and mattrin mutations; FT+/-, flaky tail mice heterozygous for filaggrin and for mattrin mutations; Il33, interleukin-33; Osm, oncostatin M; PND, postnatal day; PNE, peanut extract; WT, wild type.

Fig 5: Skin Il33, Osm, and Areg gene expression in skin-sensitized FT+/- pups. Pups were treated as in Figure 3D, but with the addition of a group receiving saline on the skin. Skin was collected at 4 h after the oral PNE gavage and analyzed by RT-qPCR for expression of (A) Osm, (B) Areg, and (C) Il33. N = 6–10/group. *p < 0.05 as compared to the saline skin-treated group. Also, for skin OSM RNA (A), the Alt/PNE/anti-Areg Ab group is not significantly different than the Alt/PNE group (p = 0.69) or the Alt/PNE/isotype Ab group (p = 0.39). Alt, Alternaria alternata extract; Areg, amphiregulin; IL33, interleukin 33; OSM, oncostatin M; PNE, peanut extract.