Figures
- Figure 1–
Discrimination by Random Forest (RF) of children with dust mite allergic rhinitis from healthy children and heat-map representation of the 61 most discriminant genes. a) Histogram showing, for each patient, the percentage of classification as a dust mite-allergic rhinitis (red) or healthy (blue) child. b) Nonsupervised, hierarchical clustering of the same patients using the set of 61 genes common to all Random Forest classifiers (see details in the Materials and methods section). Each square represents the expression level of a given gene in a given sample relative to the average expression level in controls. A red to green colour scale indicates gene expression levels above (red) or below (green) the average level of expression in healthy subjects for the same transcript. Clustering was performed using an average linkage method, using a Manhattan distance. The red colour on gene names (right) indicates genes induced in vitro by interleukin (IL)-4 and IL-13.
- Figure 2–
Discrimination by Random Forest (RF) of children with uncontrolled versus controlled asthma and heat-map representation of the 41 most discriminant genes. a) Histogram showing, for each patient, the percentage of classification as uncontrolled asthma (red) and controlled asthma (orange). b) Nonsupervised, hierarchical clustering of the same patients using the 41 probes common to all Random Forest classifiers (see Materials and methods section for details). Each square represents the expression level of a given gene in a given sample. A red to green colour scale indicates gene expression levels above (red) or below (green) the average level of the healthy controls for the same transcript. Clustering was performed using an average linkage method, using a Manhattan distance. Red-coloured gene names indicate transcripts induced by interleukin (IL)-4 and IL-13 (left) or by interferons (IFNs) (right) (corresponding to a log2(ratio) >1 in human nasal epithelial cells (HNECs)). Gene names coloured in green indicate transcripts down-regulated by IL-4 and IL-13 (left) (log2(ratio) >-1 in HNECs).
- Figure 3–
Immunohistochemical staining of sinus and bronchial biopsies. Representative immunostaining of a–c) GSDML and d–f) DUOX2 in surface epithelium and submucosal glands for healthy adults are shown. Positive immunostaining was observed for the two proteins in sinus and bronchial sections. Strong signals were also detected in submucosal glands for the different markers. Scale bars=100 μm.
- Figure 4–
Characterisation of the interferon (IFN) response. a) Measurement by real-time quantitative PCR (qPCR) of the induction of GSDML, ST8SIA4 and MX1 by IFN-α in human nasal epithelial cells (HNECs). Levels of GSDML, ST8SIA4 and MX1 were assessed in differentiated HNECs 3, 6, 12 and 24 h after stimulation with IFN-α. Measurements were performed using SYBR Green. Results are expressed as fold-change relative to the unstimulated HNECs. b) Measurement of IFNs by qPCR in patients. Levels of IFN-α1, IFN-α2, IFN-β, IFN-λ1 and IFN-λ2/3 transcripts in patients with uncontrolled asthma, patients with controlled asthma and healthy controls. Data are expressed relative to an average of 11 healthy controls. Measurements were performed using Taqman probes. Results are expressed as fold-change relative to the healthy control group. Data are presented as mean±sem. #: p<0.005 by unpaired t-test; *: p<0.05 by unpaired t-test; **: p<0.01 by unpaired t-test.
Tables
- Table 1– Subject characteristics
Group a A R C Subjects 7 6 14 11 Dust mite-allergic rhinitis Yes Yes Yes No Asthma Controlled Uncontrolled No No Age yrs 11.5±3.2 9.1±0.6 11.3±2.8 11.5±3.1 Males/females 2/5 4/2 7/7 7/4 Epithelial cells % 85.8±10.4 86.5±6.5 86.9±8.7 86.9±8.5 PMNs % 6.9±6.1 5±3.2 7.2±7.8 6.8±6.1 Lymphocytes % 7.3±10.7 8.4±6 5.5±7.3 6.3±5.6 FEV1 97.6±13.2 78.2±7.7# 99±9.4 NA FEV1/FVC 89.3±5.7 76.5±3.2# 90.4±5.2 NA FEF25–75% 103.6±17.9 56.2±9.6# 107.3±18.2 NA Data are presented as n or mean±sd, unless otherwise stated. Statistical comparisons were performed in allergic (groups a, A and R) versus healthy control subjects (group C) and in uncontrolled (group A) versus controlled (group a) asthmatics using unpaired t-tests and the Chi-squared test. PMN: polymorphonuclear cell; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; FEF25–75%: forced expiratory flow at 25–75% of FVC; NA: not available. #: p<0.005 for group A versus group a by unpaired t-test.
- Table 2– Genes differentially expressed in nasal epithelial brushings from dust mite-allergic rhinitis patients versus healthy subjects (microarray analysis)
Probe set identifier mRNA accession number UniGene identifier Gene Gene product Cytoband log2(signal) log2(allergic/healthy) p-value 8065412 NM_001898 Hs.123114 CST1 Cystatin SN 20p11.21 9.65 7.15 4.95×10−12 7971077 NM_006475 Hs.136348/Hs.664318 POSTN Periostin, osteoblast-specific factor 13q13.3 7.12 3.56 2.66×10−3 8083260 NM_001870 Hs.646 CPA3 Carboxypeptidase A3 (mast cell) 3q21–q25 6.17 2.51 5.20×10−5 7921690 NM_017625 Hs.50813 ITLN1 Intelectin 1 (galactofuranose binding) 1q22–q23.5 5.83 2.45 3.87×10−2 8065416 NM_001322 Hs.669305 CST2 Cystatin SA 20p11.21 7.97 1.87 1.06×10−3 8084657 NM_014375 Hs.81073 FETUB Fetuin B 3q27 5.78 1.77 3.63×10−3 8021635 NM_002575 Hs.594481 SERPINB2 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 18q21.3 9.20 1.64 6.97×10−3 8056222 NM_001935 Hs.368912 DPP4 Dipeptidyl-peptidase 4 (CD26) 2q24.3 6.44 1.56 6.18×10−3 7921900 NM_053282 Hs.350581 SH2D1B SH2 domain-containing 1B 1q21 5.91 1.55 3.05×10−7 8036755 NM_001828 Hs.889 CLC Charcot−Leyden crystal protein 19q13.1 4.57 1.50 2.73×10−2 7984001 NM_004751 Hs.194710 GCNT3 Glucosaminyl (N-acetyl) transferase 3, mucin type 15q21.3 8.73 1.39 8.15×10−5 8063761 NM_177980 Hs.54973 CDH26 Cadherin-like 26 20q13.2–q13.33 8.67 1.38 6.84×10−3 8089568 NM_138806 Hs.309158 CD200R1 CD200 receptor 1 3q13.2 8.90 1.35 3.90×10−5 8112668 NM_016591 Hs.272404 GCNT4 Glucosaminyl (N-acetyl) transferase 4, core 2 (β-1,6-N-acetylglucosaminyltransferase) 5q12 5.85 1.29 4.05×10−2 8070567 NM_003226 Hs.82961 TFF3 Trefoil factor 3 (intestinal) 21q22.3 9.86 1.26 2.11×10−2 7942135 NM_018043 Hs.503074 TMEM16A Transmembrane protein 16A 11q13.3 7.41 1.23 6.48×10−3 8154233 NM_014143 Hs.521989 CD274 CD274 molecule 9p24 6.75 1.23 3.84×10−5 7957458 NM_006183 Hs.80962 NTS Neurotensin 12q21 9.14 1.16 4.79×10−2 8023688 NM_002974 Hs.123035 SERPINB4 Serpin peptidase inhibitor, clade B (ovalbumin), member 4 18q21.3 8.67 1.15 3.40×10−3 8147132 NM_000067 Hs.155097 CA2 Carbonic anhydrase II 8q22 6.44 1.14 3.87×10−2 7909946 GENSCAN00000026059 NA NA NA NA 6.01 1.14 1.05×10−2 8156134 NM_006180 Hs.494312//Hs.653428 NTRK2 Neurotrophic tyrosine kinase receptor, type 2 9q22.1 5.63 1.10 5.93×10−3 8102050 NM_178833 Hs.546482 NHEDC2 Na+/H+ exchanger domain-containing 2 4q24 6.02 1.02 6.84×10−3 8089851 NM_000187 Hs.368254 HGD Homogentisate 1,2-dioxygenase (homogentisate oxidase) 3q13.33 5.71 -1.22 2.50×10−2 Genes were ranked according to decreasing log2(allergic/healthy). Only genes with a log2(ratio) >1 and a significant p-value (p<0.05) after a Benjamini–Hochberg correction for multiple tests were selected. Bold indicates genes induced in vitro with a value of log2(ratio) >1 by both interleukin (IL)-4 and IL-13 in human nasal epithelial cells. A more detailed table is provided as online supplementary table E3. NA: not available.
Supplementary material
Files in this Data Supplement:
- Supplementary tables - Tables E1-E8
- Supplementary materials and methods -
Subjects and samples
Viral respiratory tract infection detection by Real-time quantitative PCR (qPCR)
Isolation and culture of primary human nasal epithelial cells (HNECs)
Microarray analysis
Real-time quantitative PCR (qPCR)
Immunohistochemistry - Figure E1
- Figure E2
- Figure E3
- Figure E4
- Figure E5
