Online Data Supplement Immune Profile of the Airway Mucosal Lining Fluid in Neonates Skewed by Maternal Atopy Authors: Følsgaard NV, MD; Chawes BL, MD, PhD ; Rasmussen MA 2, MSci;Bischoff A, MD; Carson CG, MD; Stokholm J, MD; Pedersen L, MD; Hansel TT, MB BCh, PhD; Bønnelykke K, MD, PhD; Brix S, MSci, PhD; Bisgaard H, MD, DMsc. Methods Inclusion criterion for mothers of the COPSAC 2010 cohort Women with heart disease, endocrine disease, tuberculosis and sarcoidosis were excluded from the COPSAC 2010 study at the preliminary interview before 24 week of pregnancy. Measurements of airway inflammatory mediators After thawing, the airway epithelial lining fluid filter papers from the left and right nostrils were pooled into an Eppendorf tube, and immersed in 300 µl Milliplex Assay Buffer (Millipore, Cat no. L AB). The moist filterpapirs and assay buffer were then placed in the cup of a cellulose acetate tube filter (0.22 µm pore size) within an Eppendorf tube (Spin X Centrifuge Tube Filter, Sigma Aldrich, Cat no.cls8161), and hereafter centrifuged for 5 minutes in a cooled centrifuge at 16000g. The tube containing extracted nasal lining fluid was 1
kept on ice during aliquoting, and then stored at 80 C until analysis. The levels of IFN γ, IL 1β, IL 2, IL 4, IL 5, IL 10, IL 12p70, IL 13, TNF α, IL 8 (CXCL8), eotaxin 1 (CCL11), eotaxin 3 (CCL26), IP 10 (CXCL10), MCP 1 (CCL2), MCP 4 (CCL13), MDC (CCL22), MIP 1β (CCL4) and TARC (CCL17) in the airway epithelial lining fluid extracts were analyzed in duplicates using the Ultrasensitive Meso Scale Discovery Multi spot Human TH1/TH2 10 Plex Cytokine assay and 9 plex Chemokine assays (MesoScale Discovery, Gaithersburg, MD, USA). Assays were conducted according to standard manufacturer s protocols, except for sample/standard incubation over night at 4 C (on shaker) and the use of an eleven point standard curve from 5000 to 0.31 pg/ml for cytokines and at 20000 to 1.25 pg/ml for chemokine determinations. Samples were read using the Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, MD, USA). The lower limit of detection was determined to be (based on mean signals from blanks + 3*SD): IFN γ: 4.09, IL 1β: 1.48, IL 2: 1.48, IL 4: 1.05, IL 5: 1.24, IL 10: 1.00, IL 12p70: 1.44, IL 13: 2.71, TNF α: 0.61, IL 8: 3.0, eotaxin 1: 22, eotaxin 3: 45, IP 10: 31, MCP 1: 16, MCP 4: 2.8, MDC: 25, MIP 1β: 4.3 and TARC: 7.2; all in pg/ml. To see a video of the method for sampling mucosal lining fluid with filter papir on 4 week old baby please press, please access the table of contents for this issue of the AJRCCM. A link will be available there. The infant in the video is administered sugar water (20 % dilution) during sampling to inhibit sneeze reflex and soothe any discomfort. Apgar score: The Apgar score is designed to assess infants' condition immediately after birth. The score is reported at 1 and 5 minutes after birth as a cumulative ranking of five clinical signs heart 2
rate, respiratory effort, muscle tone, reflex activity, and color. Each assigned a rating of 0, 1, or 2 with lower number corresponding to poorer condition, thereby ranging the childs condition at birth from 0 to 10. The Apgar score provides a convenient tool for assessing the newborn infant at birth and the response to resuscitation. However, the Apgar test is not designed to make long term predictions on the child's health. 1 Statistics: Principal component analysis is a statistical method where we look at the variation of data in a multi dimensional space. We use PCA to extract underlying latent variables called principal components that describes the systematic part of the variation across the 18 mediators in a few orthogonal (uncorrelated) components. Each component comprise of a score vector corresponding to the samples and a loading vector corresponding to the variables. The score vector can be interpreted as a pseudo variable, and reflects the distribution of samples across all variables. Principal Component 1 describes the greatest part of the variation of the data, Principal Component 2 the next greatest and so forth. The loadings reflects the correlation structure of the variables, and hence can be interpreted alone for biological patterns across mediators, and in connection with the scores for details on which samples that exhibit a certain pattern. The correlation structure is visualized by scatter plots of loadings for principal component 1 and 2. Scatter plots of the score vectors for principal component 1 versus 2 were used to investigate differences in overall cytokine/chemokine response between the children with or without atopic mothers. Score vector for component 1 through 4 was used assimilated to the original variables (pseudo variables) when testing towards outcomes. 3
Results: Table E1a and E1b: Baseline characteristics of neonate of atopic fathers (with or without atopic mother) vs. non atopic fathers (with or without atopic mothers) (a) and neonates of atopic mothers (without atopic fathers) vs. atopic fathers (without atopic mothers) (b). Table E2: Baseline levels of cytokines and chemokines Table E3: Association between mediator levels and parental atopic status in the study group. Figure E1: PCA loading Plot for mediators vs. endpoints. X axis shows that 66.5 % of the material is described in PC1 and only a further 8 % in PC2 (Yaxis) illustrating that all variables are strongly correlated, since the first component describes 66.5 % of the total variation. Therefore, the statistically significant univariate associations mainly reflect one general underlying inhibiting tendency in the children of atopic mothers. Figure E2. Separation plot for neonates with atopic fathers (with or without atopic mothers) (Atopic F +) vs. non atopic fathers (with or without atopic mothers)(atopic F ). Ellipsoids are centered at the mean and rotated according to correlation with latent variables (PC1 and PC2) using standard deviation as semi axis. For separation in PC1 (p = 0.44 Mann Whitney U test, p = 0.59 t test) and PC2 (p = 0.53 MWU, p = 0.32 t test). Figure E3: Separation plot for neonates with non atopic fathers (AF ) and atopic mothers (AM+)(n=93) vs. atopic fathers (AF+) and non atopic mothers (AM ) (n=68). Ellipsoids are centered at the mean and rotated according to correlation with latent variables (PC1 and PC2) using standard deviation as semi axis. For separation in PC1 (p = 0.02 Mann Whitney U test, p = 0.02 t test) and PC2 (p = 0.34 MWU, p =0.13 t test). 4
Video of the sampling method: This video shows the sampling of mucosal lining fluid on a 4 week old baby. This is done by inserting 3x15 mm strips of filter paper (Accuwik Ultra, fibrous hydroxylatedpolyester sheets) onto the anterior part of the inferior nasal turbinate of both nostrils for 2 minutes. The infant in the video is administered sugar water (20 % dilution) during sampling to inhibit sneeze reflex and soothe any discomfort. (This video is available through the table of contents of this issue of the AJRCCM. Please see the link provided there). Reference List 1. Robert M.Kliegman. 2007. Nelson Textbook of Pediatrics, 18th ed. Saunders. 5
Table E1a: Baseline demographics. Atopic fathers (with or without atopic mothers) vs. non atopic fathers (with or without atopic mothers) Baseline All Atopic fathers Non atopic P value %(N) %(N) fathers %(N) 309 47% (141)* 53% (159) Pre natal exposure Atopic mothers 56% (168) 52% (73) 60% (95) 0.16 Caucasian 96% (286) 94% (132) 97% (154) 0.10 Household income high** 36% (107) 36% (50) 36% (57) 0.22 Antibiotics in 3 rd trimester 18% (53) 20% (28) 16% (25) 0.35 Furred animals during pregnancy 33% (100) 32% (45) 35% (55) 0.68 Maternal smoking during pregnancy 5% (15) 5% (7) 5% (8) 0.97 Alcohol >1 unit/week during 3 rd trimester 6% (18) 4% (6) 8% (12) 0.22 Post natal exposure Gestational age*** 5% (16) 8% (11) 3% (5) 0.10 Low birthweight (u. 2500g) 1%(3) 2%(2) 1%(1) 0.48 Male 50% (151) 53% (74) 48% (77) 0.48 Apgar score <7 at 1 min. 4% (13) 4% (6) 5% (7) 0.25 Caesarian section 11% (34) 9% (12) 14% (22) 0.17 Older sibling 49% (147) 49% (69) 49% (78) 0.98 Exclusive breastfeeding until study day 90% (272) 91% (129) 90% (143) 0.71 * Missing data on fathers atopic status for 9 individuals.** High; above 110.000 euro a year.***before 37 weeks. Table E1b: Baseline demographics. Atopic mothers (non atopic fathers) vs. atopic fathers (non atopic mothers) Baseline All Atopic mothers Atopic fathers P value %(N) (non atopic fathers) (non atopic mothers) %(N) %(N) 163 58% (95) 42% (68) Pre natal exposure Caucasian 96% (156) 99% (93) 93% (63) 0.10 Household income high* 38% (62) 34% (32) 44% (30) 0.24 Antibiotics in 3 rd trimester 16% (28) 19% (13) 16% (15) 0.58 Furred animals during pregnancy 29% (48) 32% (30) 26% (18) 0.85 Maternal smoking during pregnancy 4% (7) 3% (3) 6% (4) 0.39 Alcohol >1 unit/week during 3 rd trimester 6% (10) 7% (7) 4% (3) 0.43 Post natal exposure Gestational age** 6% (9) 3% (3) 9% (6) 0.12 Low birthweight (u. 2500g) 1%(2) 1%(1) 1%(2) 0.77 Male 45% (73) 43% (41) 47% (32) 0.62 Apgar score <7 at 1 min. 6% (9) 6% (6) 5% (3) 0.31 Caesarian section 13% (21) 15% (14) 10% (7) 0.15 Older sibling 49% (80) 48% (46) 50% (34) 0.84 Exclusive breastfeeding until study day 89% (175) 88% (84) 90% (61) 0.50 * High; above 110.000 euro a year.**before 37 weeks.
Table E2: Baseline levels of cytokines and chemokines Detectable Undetectable levels /Missing data Mean (95% CI) concentration in samples with detectable levels (pg/ml) IFN γ 300 9 5.0 (4.1,5.5) IL 10 306 3 12.2 (9.9,14.9) IL 12p70 308 1 3.0(2.4,3.3) IL 13 307 2 8.1(6.6,9.0) IL 1β 309 0 81.45(66.7,99.5) IL 2 309 0 9.9(9.0,11.1) IL 4 289 20 0.9(0.8,1.1) IL 5 309 0 1.6(1.3,1.8) IL 8 309 0 2697(2440,3294) TNF α 307 2 18.17(14.8,22.2) Eotaxin 1 309 0 49.4(44.7,55.3) Eotaxin 3 285 24 49.4(44.7,55.1) IP 10 309 0 492.7(407.5,601.8) MCP 1 309 0 90.0(81.4,99.5) MCP 4 309 0 8.00(7.5,9.0) MDC 285 24 90.0(81.5,99.5) MIP 1β 309 0 99.5(81.5,121.5) TARC 309 0 11.0(9.9,11.0)
Table E3: Associations between mediator levels and parental atopic status in the study group. Neonates with atopic fathers (with or without atopic mothers) compared to non atopic fathers (with or without atopic mothers) Neonates with atopic mothers (with or without atopic fathers) compared to neonates of non atopic mothers (with or without atopic fathers) Neonates of atopic mothers and nonatopic fathers compared to neonates of atopic fathers and non atopic mothers Ratio (95% CI) P value Ratio (95% CI) P value Ratio (95% CI) P value IFN γ 1.2 (0.8;1.8) P=0.37 0.6 (0.34;0.9) P= 0.02 0.5 (0.3;0.9) P= 0.4 IL 10 0.9 (0.6;1.5) P= 0.95 0.8 (0.6;1.3) P= 0.44 0.8 (0.5;1.3) P= 0.04 IL 12P70 0.9 (0.7;1.2) P= 0.61 0.8 (0.6;1.0) P= 0.07 0.8 (0.6;1.1) P= 0.01 IL 13 0.9 (0.8;1.2) P= 0.87 0.8 (0.6;0.9) P= 0.03 0.8 (0.6;1.1) P= 0.2 IL 1b 1.4 (0.9;2.2) P= 0.19 0.7 (0.4;0.9) P= 0.04 0.4 (0.2;0.8) P= 0.1 IL 2 1.1 (0.8;1.4) P=0.55 0.8 (0.6;0.9) P= 0.04 0.7 (0.5;0.9) P= 0.01 IL 4 1.0 (0.6;1.7)P= 0.95 0.6 (0.3;0.9) P= 0.02 0.5 (0.3;0.8) P= 0.004 IL 5 1.0 (0.7;1.3) P= 0.99 0.9 (0.7;1.2) P= 0.42 0.8 (0.6;1.2) P= 0.3 IL 8 1.2 (0.9;1.6) P=0.34 0.7 (0.5;0.9) P= 0.01 0.6 (0.4;0.9) P=0.06 TNF α 1.1 (0.7;1.6) P= 0.67 0.6 (0.4;0.9) P= 0.02 0.6 (0.4;1.0) P= 0.006 Eotaxin 1 1.2 (0.9;1.4) P= 0.08 0.8 (0.7;0.9) P= 0.04 0.7 (0.5;0.9) P= 0.15 Eotaxin 3 0.7 (0.5;1.1) P= 0.09 0.6 (0.4;0.9) P= 0.006 0.8 (0.6;1.1) P= 0.007 IP 10 0.9 (0.6;1.3) P=0.72 0.6 (0.4;0.9) P= 0.005 0.6 (0.4;1.1) P= 0.07 MCP 1 1.3 (1.1;1.7) P= 0.02 0.8 (0.6;0.9) P= 0.03 0.6 (0.4;0.8) P= 0.0006 MCP 4 1.1 (0.9;1.3) P=0.31 0.9 (0.7;0.9) P= 0.05 0.8 (0.6;0.9) P= 0.02 MDC 0.8 (0.6;1.1) P= 0.22 0.7 (0.5;0.9) P= 0.02 0.8 (0.6;1.0) P= 0.06 MIP 1β 1.2 (0.8;1.8) P=0.33 0.8 (0.5;1.2) P= 0.27 0.6 (0.4;1.1) P= 0.1 TARC 1.1 (0.9;1.3) P=0.17 0.9 (0.8;1.0) P= 0.06 0.8 (0.6;0.9) P= 0.02
Figure E1: PCA loading Plot for mediators vs. endpoints. X axis shows that 66.5 % of the material is described in PC1 and only a further 8 % in PC2 (Yaxis) illustrating that all variables are strongly correlated, since the first component describes 66.5 % of the total variation. Therefore, the statistically significant univariate associations mainly reflect one general underlying inhibiting tendency in the children of atopic mothers.
Figure E2. Separation plot for neonates with atopic fathers (with or without atopic mothers) (Atopic F +) vs. non atopic fathers (with or without atopic mothers)(atopic F ). Ellipsoids are centered at the mean and rotated according to correlation with latent variables (PC1 and PC2) using standard deviation as semi axis. For separation in PC1 (p = 0.44 Mann Whitney U test, p = 0.59 t test) and PC2 (p = 0.53 MWU, p = 0.32 t test). 4 Atopic F - Atopic F + 3 2 1 PC 2 0-1 -2-3 -4-10 -8-6 -4-2 0 2 4 6 PC 1
Figure E3: Separation plot for neonates with non atopic fathers (AF ) and atopic mothers (AM+)(n=93) vs. atopic fathers (AF+) and non atopic mothers (AM ) (n=68). Ellipsoids are centered at the mean and rotated according to correlation with latent variables (PC1 and PC2) using standard deviation as semi axis. For separation in PC1 (p = 0.02 Mann Whitney U test, p = 0.02 t test) and PC2 (p = 0.34 MWU, p =0.13 t test). 4 3 AM + AF - AM - AF + the rest 2 1 PC 2 0-1 -2-3 -4-10 -8-6 -4-2 0 2 4 6 PC 1