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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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