Research ArticleAsthma

Distinct immune phenotypes in infants developing asthma during childhood

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Science Translational Medicine  05 Feb 2020:
Vol. 12, Issue 529, eaaw0258
DOI: 10.1126/scitranslmed.aaw0258
  • Fig. 1 Comprehensive analysis of circulating innate cell and T cell function in 18-month-old infants.

    (A) Study outline. Blood for immune analysis was drawn at 18 months of age, and disease development was evaluated throughout the first 6 years of life. (B) Analytical overview. The 18 listed immune cell subsets were detected by multiparametric flow cytometry in freshly collected whole blood (n = 552). (C) In parallel, diluted whole blood was incubated 24 hours with media only (spontaneous release) or the indicated innate and T cell activating ligands, followed by measurement of cytokines and chemokines within harvested cell-free supernatants using electrochemoluminescence-based immunoassays (n = 567). Concentrations of the pretitrated ligands are available in Materials and Methods. The detected cytokines and chemokines belong to five immune response types, here assigned a specific color-code used in subsequent figures of the manuscript for ease of explanation.

  • Fig. 2 Circulating immune cell frequencies and response profiles in whole blood of 18-month-old infants.

    (A) Relative frequencies of the 18 immune cell subsets in whole blood (n = 552). (B) Concentrations of cytokines and chemokines from 24 hours unstimulated (spontaneous release) whole blood. (C) Polar charts illustrating the mean fold change of cytokine and chemokine release in innate ligand stimulated versus unstimulated whole blood. For stimulation of innate immune cells, blood was stimulated to activate TLR3 [viral double-stranded RNA (dsRNA)], TLR7/8 (viral ssRNA), TLR4 (bacterial LPS), NOD2 (bacterial peptidoglycan), and NLRP3 (alum with low-dose LPS). For NLRP3, the mean fold change was calculated on the basis of addition or not of alum to a low-dose LPS control culture (5 ng/ml). For T cell stimulation, blood was stimulated with SEB and HDMAPP to activate αβ T cells and γδ T cells, respectively. (B and C) Color codes correspond to the type-response designations from Fig. 1C. N = 567.

  • Fig. 3 Sex-related variations in circulating immune cell numbers and the functional cytokine response after innate ligand stimulation of blood immune cells in 18-month-old infants.

    Linear models were applied to compare concentrations of circulating immune cells (A) and cytokine release upon innate ligand stimulation (B) with female sex as the outcome. Positive estimate size (red) corresponds to higher concentrations within girls, and negative estimate size (blue) corresponds to higher concentrations within boys. The statistical significance is reflected in the size of the dot and is reported as false discovery rate (FDR)–adjusted P values. N = 541.

  • Fig. 4 Ligand-dependent associations between immune cell subsets and the functional response pattern in blood of 18-month-old infants.

    The cell-to-cytokine covariation matrices per activating stimuli from Fig. 2, visualized as SCCs between relative cell frequencies and delta concentrations of released cytokine from stimulated versus unstimulated whole blood. Whole blood was stimulated to activate TLR3 (viral dsRNA), TLR7/8 (viral ssRNA), TLR4 (bacterial LPS), NOD2 (bacterial peptidoglycan), and NLRP3 (alum in concert with low-dose LPS). Unstimulated controls were added media alone. NLRP3 data were based on alum + low LPS stimulation minus the low LPS stimulation control. SCC ranges from −0.5 to 0.5 (legend at the bottom) and are only plotted if P < 0.01. Cytokines are color-coded at the top to match the designations of response types as in Fig. 1C. N = 541.

  • Fig. 5 Great heterogeneity within innate ligand stimulated immune responses in blood of 18-month-old infants.

    For each of the five innate ligands, we selected cytokines for which the cell-to-cytokine SCC was above 0.25 and subgrouped the response profiles based on hierarchical clustering. The color code for the chosen cytokines corresponds to the type-response designations from Fig. 1C. Data are z-score normalized per cytokine and plotted as the average score within each cluster. The identified clusters for each ligand are named by C and a number. The overlap across innate stimuli and response profile (cluster) for each individual is provided in data file S1. The percentage of infants in a given cluster is given by the width of the cluster and printed below each cluster. N = 541.

  • Fig. 6 A distinct antiviral innate response profile in 18-month-old infants enhances the risk of developing transient childhood asthma.

    Asthma development was followed longitudinally from birth to 6 years of age in the research clinic and defined as either transient or persistent asthma at 6 years of age. (A) The dot plot displays the relative risk of transient asthma development in infants within the given cluster versus the risk of transient asthma in the remaining infants. An encircled dot indicates the statistically significant association given in the text with Padj < 0.05. (B) Cox proportional hazards regression analysis of transient asthma development until 6 years of age in infants within the TLR7/8-C7 cluster. Percentage of infants in each cluster is given in Fig. 5. The Padj is determined by Benjamini-Hochberg FDR correction. (C) Relative prevalence of blood immune cells at 18 months of age in infants developing transient childhood asthma versus non-asthmatic children at 6 years of age. Cells were identified on the basis of flow cytometry of freshly collected blood and gated as illustrated in fig. S2. N = 541.

  • Fig. 7 IL-5– and IL-13–based T cell profile at 18 months of age associates with development of persistent childhood asthma.

    (A) Stimulated αβ T cell response cytokines were selected and used for subgrouping of response profiles based on hierarchical clustering (z score–normalized per cytokine; fig. S9), resulting in six T cell subgroups. The percentage of infants in a given cluster is given by the dimension of the cluster and printed below each cluster. (B) The dot plot displays the prevalence of the indicated asthma phenotype (overall asthma until 6 years of age, persistent asthma at 6 years of age, transient asthma until 6 years of age) of 18-month-old infants within the indicated T cell cluster (x axis) as compared to remaining infants. Data are shown as ratios calculated as the prevalence of infants with disease within the indicated cluster versus the disease prevalence within remaining infants. An encircled dot indicates the statistically significant association given in the text with Padj < 0.05. (C) Cox proportional hazards regression analysis of persistent asthma development (0 to 6 years) within the two IL-5– and IL-13–enriched clusters at 18 months of age compared to remaining infants. The Padj is determined by Benjamini-Hochberg FDR correction. N = 541.

  • Table 1 Genetic and environmental determinants of the at-risk immune cluster for persistent asthma.

    Logistic regression analyses of genetic and environmental risk determinants on children in the at-risk immune clusters, αβTCR TH2 + mixed, versus remaining children of the cohort. CI, confidence interval.

    αβTCR TH2 + mixedRemainingαβTCR TH2 + mixed/remaining
    OR [CI]P value
    N169366
    Child
      Sex, male, % (N)54% (91)49% (179)0.72 [0.46–1.13]0.15
      17q21, % (N)*28% (42)33% (108)0.88 [0.52–1.44]0.61
      Any airway bacteria at 1 month, % (N)38% (63)25% (89)1.87 [1.26–2.78]0.0020
    Atopy
      Maternal asthma, % (N)35% (59)27% (100)1.43 [0.96–2.11]0.076
      Paternal asthma, % (N)27% (44)23% (81)1.24 [0.81–1.89]0.32
      Maternal sensitization, % (N)40% (67)35% (126)1.25 [0.85–1.81]0.25
      Paternal sensitization, % (N)51% (82)45% (156)1.27 [0.87–1.85]0.22
      Child sensitization, 6 or 18 months, % (N)8% (13)11% (38)0.70 [0.35–1.31]0.27
      Childhood eczema, 0–6 years, % (N)35% (59)30% (109)1.26 [0.86–1.86]0.24
      Leukotriene receptor antagonist, % (N)10% (11)5% (12)2.13 [0.90–5.02]0.084
      Inhaled corticosteroid treatment, % (N)23% (26)16% (41)1.52 [0.87–2.62]0.14
    Pregnancy
      Smoking in pregnancy, % (N)5% (9)8% (31)0.61 [0.27–1.26]0.19
      Cat or dog in pregnancy, % (N)31% (52)33% (122)0.89 [0.60–1.31]0.56
      Antibiotics in pregnancy, % (N)40% (67)34% (125)1.26 [0.86–1.84]0.23
      Fish oil supplementation, % (N)51% (87)48% (175)1.16 [0.80–1.67]0.43
      Vitamin D supplementation, % (N)52% (80)49% (160)1.14 [0.77–1.67]0.51
    Birth
      Term birth >37 weeks, % (N)98% (165)96% (351)1.76 [0.63–6.26]0.30
      Primiparity, % (N)41% (69)49% (178)0.73 [0.50–1.05]0.092
      APGAR score 5 min >9, % (N)97% (162)94% (341)1.90 [0.75–5.79]0.18
      Caesarean section, % (N)14% (22)19% (69)0.65 [0.38–1.08]0.10
      Season of birth, fall/winter, % (N)58% (98)55% (200)1.15 [0.79–1.66]0.47
      Maternal age at birth, years, mean (SD)32.7 (4.4)32.2 (4.2)1.03 [0.99–1.08]0.17

    *% homozygous for the risk allele, variant RS2305480.

    †Presence of Moraxella catarrhalis, Haemophilus influenzae or Streptococcus pneumoniae.

    ‡14 days up to sample date.

    Supplementary Materials

    • stm.sciencemag.org/cgi/content/full/12/529/eaaw0258/DC1

      Materials and Methods

      Fig. S1. Overview of blood sampling at 18 months of age.

      Fig. S2. Gating strategy for enumeration of immune cells by flow cytometry.

      Fig. S3. Correlations between relative cell frequencies and absolute cell counts.

      Fig. S4. Multivariate analysis of cytokine profiles from innate ligand-stimulated blood collected at 18 months of age.

      Fig. S5. Hierarchical clustering of the functional response profile to innate ligands in blood immune cells from 18-month-old infants.

      Fig. S6. Distinct antimicrobial innate response profiles in infants associate to overall risk of childhood asthma.

      Fig. S7. Reduced risk of transient childhood asthma in infants within the NLRP3-C2 at 18 months of age.

      Fig. S8. Early immune phenotypes in infants that develop persistent childhood asthma.

      Fig. S9. Hierarchical clustering of the functional response profile in blood αβ T cells from 18-month-old infants.

      Table S1. Demographic data of study cohort.

      Table S2. Relative frequencies of immune cell subsets in whole blood at 18 months of age.

      Table S3. Whole-blood concentration of immune cell subsets in 18-month-old infants.

      Table S4. Concentrations of cytokines and chemokines after 24 hours incubation with media only (unstimulated, i.e., spontaneous release) or ligands targeting the indicated receptors.

      Table S5. Genetic and environmental determinants of TLR3 low responders.

      Table S6. Genetic and environmental determinants of TLR4 low responders.

      Table S7. Genetic and environmental determinants of TLR7/8 low responders.

      Table S8. Genetic and environmental determinants of NOD2 low responders.

      Table S9. Genetic and environmental determinants of NLRP3 low responders.

      Table S10. Genetic and environmental determinants of αβTCR low responders.

      Table S11. Genetic and environmental determinants of transient and persistent asthma.

      Table S12. Genetic and environmental determinants of at-risk TLR7/8-C7 immune cluster for transient asthma.

      Data file S1. Intra-individual matrix comparing the response type to the antiviral and antibacterial ligands and the T cell response type.

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Overview of blood sampling at 18 months of age.
      • Fig. S2. Gating strategy for enumeration of immune cells by flow cytometry.
      • Fig. S3. Correlations between relative cell frequencies and absolute cell counts.
      • Fig. S4. Multivariate analysis of cytokine profiles from innate ligand-stimulated blood collected at 18 months of age.
      • Fig. S5. Hierarchical clustering of the functional response profile to innate ligands in blood immune cells from 18-month-old infants.
      • Fig. S6. Distinct antimicrobial innate response profiles in infants associate to overall risk of childhood asthma.
      • Fig. S7. Reduced risk of transient childhood asthma in infants within the NLRP3-C2 at 18 months of age.
      • Fig. S8. Early immune phenotypes in infants that develop persistent childhood asthma.
      • Fig. S9. Hierarchical clustering of the functional response profile in blood αβ T cells from 18-month-old infants.
      • Table S1. Demographic data of study cohort.
      • Table S2. Relative frequencies of immune cell subsets in whole blood at 18 months of age.
      • Table S3. Whole-blood concentration of immune cell subsets in 18-month-old infants.
      • Table S4. Concentrations of cytokines and chemokines after 24 hours incubation with media only (unstimulated, i.e., spontaneous release) or ligands targeting the indicated receptors.
      • Table S5. Genetic and environmental determinants of TLR3 low responders.
      • Table S6. Genetic and environmental determinants of TLR4 low responders.
      • Table S7. Genetic and environmental determinants of TLR7/8 low responders.
      • Table S8. Genetic and environmental determinants of NOD2 low responders.
      • Table S9. Genetic and environmental determinants of NLRP3 low responders.
      • Table S10. Genetic and environmental determinants of αβTCR low responders.
      • Table S11. Genetic and environmental determinants of transient and persistent asthma.
      • Table S12. Genetic and environmental determinants of at-risk TLR7/8-C7 immune cluster for transient asthma.
      • Legend for data file S1

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Data file S1 (Microsoft Excel format). Intra-individual matrix comparing the response type to the antiviral and antibacterial ligands and the T cell response type.

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