Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata
Article first published online: 20 FEB 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 4, pages 907–917, April 2013
How to Cite
Denis, O., Vincent, M., Havaux, X., De Prins, S., Treutens, G. and Huygen, K. (2013), Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata. Eur. J. Immunol., 43: 907–917. doi: 10.1002/eji.201242630
- Issue published online: 16 APR 2013
- Article first published online: 20 FEB 2013
- Accepted manuscript online: 14 JAN 2013 09:04AM EST
- Manuscript Accepted: 8 JAN 2013
- Manuscript Revised: 26 NOV 2012
- Manuscript Received: 19 APR 2012
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Supplemental Table 1: Primers used for the qRT-PCR
Supplementary Fig. 1: Cytokine/chemokine mRNA expression after stimulation with native or heat-treated Alternaria extracts. BALB/c mice were instilled during 3 days (A) or 4 weeks (B) with PBS, untreated or denatured Alternaria extract. Total RNA was extracted from the whole lungs and transcriptional levels of the indicated mediators were analyzed by qRT-PCR. Results are shown as mean ± SD of 5 individual mice. NS= non significant, * p<0.05, ** p<0.01; two-tailed Mann-Whitney U-test.
Supplementary Fig. 2: Histopathological changes in the lungs of mice chronically instilled with native or denatured Alternaria extract. BALB/c mice were given i.n. instillations of PBS, untreated or denaturated Alternaria extract for 5 weeks. Lung sections were prepared and stained with haematoxylineosin- safran (HES), Periodic Acid Schiff (PAS) and Masson trichrome (MT).
Supplementary Fig. 4: Quantification of the LPS content in the Alternaria extract (50 μg/mL), in an OVA solution (50 μg/mL) or in the same OVA solution spiked with 2 μg/mL of LPS (A). The LPS concentration in extract was 0.804 EU/mL hence 0.0804 EU LPS were instilled each time resulting in a total amount of 1.04 EU instilled during the 5-week period. An OVA solution was prepared in PBS in conditions similar to those used for the preparation of the extracts. An OVA solution spiked with 2 μg/mL of LPS was also prepared. Measurement of LPS content of these preparations detected a concentration of 0.978 EU/mL in the unspiked OVA solution (0.0978 EU/instillation leading to a total amount of 1.271 EU instilled during the 5-week period) and a concentration of 3200 EU/mL for the spiked OVA+LPS solution (320 EU/instillation, 4200 EU/5 weeks). Mice were chronically instilled with the Alternaria extract, with OVA or with OVA + LPS (5μg ext or ova in each instillation) during 5 weeks and the allergic immune response was analyzed. Extract instillation induced high IgE serum levels but IgE levels remained low after OVA or OVA + LPS instillation (B). Extract instillations also stimulated specific IgG1 production. OVA instillations induced minimal IgG1 levels in the serum but IgG1 levels were highly increased when OVA+ LPS was used (C). Results are shown as mean ± SEM of eight mice. In vitro specific re-stimulation of splenocytes from mice instilled with the OVA or the OVA + LPS did not show any secretion of IL-13 in the supernatants, in contrast to the splenocytes from Alternaria extract instilled mice which strongly produced IL-13 upon in vitro re-stimulation (D). Results are shown as mean ± SD of four mice. Alternaria extract instillations led to a lung inflammation characterized by the recruitment of macrophages, neutrophils, lymphocytes and high numbers of eosinophils, whereas OVA + LPS instillations induced only a minimal inflammation with macrophages and neutrophils and OVA instillations did not induce any lung inflammation (E). Finally, in accordance with the results obtained in vitro, IL-13 was detected in the BALF of mice treated with the Alternaria extract but not with OVA or OVA+LPS (F). Results are shown as mean ± SD of seven to eight mice. ** p<0.01; two-tailed Mann–Whitney U-test.
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