Supplementary methods
Collection and analysis of bronchoalveolar lavage (BAL) and lung cells.
BAL was performed by flushing the airways three times with 1 ml PBS. Total BAL
cells were counted using a Coulter Counter (IG Instruments) and spun onto glass
slides using a Cytospin 2 (Shandon Southern Products, Ltd.). Cells were then stained
with Diff Quick staining set (Siemens-Dade Behring). Percentages of eosinophils,
macrophages, lymphocytes and neutrophils were determined microscopically using
standard morphological and cytochemical criteria. Lungs were perfused with 10 mL
PBS and digested in medium supplemented with 2 mg/ml Collagenase IV.
Afterwards, a 30% Percoll gradient was applied to the cells to isolate lung leukocytes.
Lung histology
Mice were sacrificed, the trachea was cannulated and the lung inflated with 1 mL 4%
neutral buffered formalin. The lung was then removed, collected in formalin and
processed for Hematoxylin and Eosin (H&E) and Periodic acid-Schiff (PAS) staining.
Histological sections were evaluated according to cellular infiltration and goblet cell
hyperplasia.
Antibodies and FACS analysis
Surface staining was carried out in PBS/0.1% BSA. Monoclonal Ab recognizing
CD4, CD25, and CD11c were purchased from eBioscience; anti-CD11b, -CD40, -IAb , --TCR mAb were purchased from BD Pharmingen. For cytokine staining, BAL
cells were stimulated with PMA/ionomycin for 4 h, with the addition of Monensin
(Sigma-Aldrich). In general, for intracellular staining, cells were fixed with 2%
paraformaldehyde and subsequently incubated in permeabilization buffer (0.5%
saponin/PBS/0.5% BSA) containing anti-IL-4, -IFN-, -FoxP3, -IL-17 (all
eBioscience) or anti-IL-5 mAb (BD Pharmingen). After washing, cells were
resuspended in PBS/0.1% BSA and analyzed by flow cytometry (FACS Calibur; BD
Biosciences) and FlowJo software (Tree Star). Sorting of lung cell populations was
performed on a FACS Aria (BD Biosciences).
ELISA
Capture anti-IL-5 antibody and biotin-conjugated anti-IL-5 detection antibody were
purchased from BD Pharmingen. Alkaline phosphatase-labeled streptavidin (Southern
Biotechnology) and substrate p-nitrophenyl phosphate (Sigma-Aldrich) were used.
OD was determined at 405 nm using a SpectraMax spectrophotometer (Bucher
Biotech). For IL-13 detection, IL-13 ELISA Ready-SET-go! (eBioscience) was used.
Measurement of Airway Responsiveness
On day 3 after i.n. challenge with OVA, mice were placed in individual, unrestrained
whole body plethysmograph chambers (Buxco Electronics). Airway responsiveness
was assessed in mice by inducing airflow obstruction with aerosolized methacholine
chloride (MetCh). Pulmonary airflow obstruction was assessed by measuring PenH
using BioSystem XA software (Buxco Electronics).
Supplementary results
Signaling through NOD1 and NOD2 does not contribute to suppression of
inflammation.
In addition to TLR4, we investigated the role of the intracellular patternrecognition receptor NOD1 and its downstream signaling molecule RIP2 in inhibiting
AAI. Notably, NOD1 recognizes cell wall-components of E.coli, and has been listed
as a susceptibility gene in the development of asthma 1; RIP2 has been shown to be
essential in mediating signaling events downstream of NOD1 as well as NOD2 2. We
exposed NOD1 and Rip2 deficient mice to E.coli followed by OVA sensitization and
challenge; in the absence of NOD1 and Rip2, E.coli treatment still impaired
eosinophil influx in the airways, suggesting that NOD1 and NOD2-dependent
inflammatory pathways do not contribute to the inhibition of AAI (Supplementary
Fig. 3).
Mice treated with E.coli are still able to respond to TLR4 activation.
We sought to test whether the E.coli treatment led to a desensitization of
TLR4 signaling, which could influence the induction of the inflammation 3. Control
and E.coli-treated mice were challenged intranasally with increasing doses of LPS
and the extent of inflammation was analyzed after 24h. The resulting recruitment of
neutrophils to the airways was in fact greater in the previously E. coli-treated mice,
ruling out a possible role of TLR4-desensitization in mediating AAI suppression in
our model (Supplementary Fig. 4).
Supplementary Figure Legends
Supplementary Fig. 1: 107 CFU E.coli were administered intranasally to C57BL/6 mice: neutrophil
influx in the BAL was determined at different time points after administration by differential cell
counts (A). The presence of live bacteria in the lung was determined by plating single cell suspensions
on agar plates (B). n.d., not detectable.
Supplementary Fig. 2: C57BL/6 mice were treated with E.coli 3 weeks prior to immunization: airway
hyperresponsiveness (left), eosinophil numbers (middle) and proportion of IL-4- or IL-5-producing
CD4 T cells (right) were determined as described in Fig.1. Results are presented as mean  SD. *, P <
0.05; statistically significant differences. Experiments were repeated 2 times with 3-4 mice per group.
Supplementary Fig. 3: Eosinophil numbers in the BAL of C57BL/6, Nod1 and Rip2 deficient mice
were determined 4 days after challenge with OVA. Results are presented as mean  SD. *, P < 0.05;
statistically significant differences. Experiments were repeated 2 times with 4 mice per group.
Supplementary Fig. 4: Non-treated (control) and E.coli-treated mice were sensitized with OVA in
alum and challenged intranasally 7 days later with the indicated doses of LPS. The number of
neutrophils infiltrating the lung was determined by differential cell counts 24 h after the challenge.
Results represent values from single animals or are presented as mean  SD. *, P < 0.05; statistically
significant differences. Experiments were repeated 2 times with 3-4 mice per group.
Supplementary References
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Eder, W., et al. Association between exposure to farming, allergies and
genetic variation in CARD4/NOD1. Allergy 61, 1117-1124 (2006).
Nembrini, C., et al. The kinase activity of Rip2 determines its stability and
consequently Nod1- and Nod2-mediated immune responses. J Biol Chem 284,
19183-19188 (2009).
Didierlaurent, A., et al. Sustained desensitization to bacterial Toll-like
receptor ligands after resolution of respiratory influenza infection. J Exp Med
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