Elevated faecal 12,13-diHOME concentration in neonates at high risk for asthma is produced by gut bacteria and impedes immune tolerance

Abstract

Neonates at risk of childhood atopy and asthma exhibit perturbation of the gut microbiome, metabolic dysfunction and increased concentrations of 12,13-diHOME in their faeces. However, the mechanism, source and contribution of this lipid to allergic inflammation remain unknown. Here, we show that intra-abdominal treatment of mice with 12,13-diHOME increased pulmonary inflammation and decreased the number of regulatory T (T_reg) cells in the lungs. Treatment of human dendritic cells with 12,13-diHOME altered expression of PPARγ-regulated genes and reduced anti-inflammatory cytokine secretion and the number of T_reg cells in vitro. Shotgun metagenomic sequencing of neonatal faeces indicated that bacterial epoxide hydrolase (EH) genes are more abundant in the gut microbiome of neonates who develop atopy and/or asthma during childhood. Three of these bacterial EH genes (3EH) specifically produce 12,13-diHOME, and treatment of mice with bacterial strains expressing 3EH caused a decrease in the number of lung T_reg cells in an allergen challenge model. In two small birth cohorts, an increase in the copy number of 3EH or the concentration of 12,13-diHOME in the faeces of neonates was found to be associated with an increased probability of developing atopy, eczema or asthma during childhood. Our data indicate that elevated 12,13-diHOME concentrations impede immune tolerance and may be produced by bacterial EHs in the neonatal gut, offering a mechanistic link between perturbation of the gut microbiome during early life and atopy and asthma during childhood.

Fig. 1 |. Peritoneal treatment with 12,13-diHOME exacerbates lung inflammation in mice challenged with CRA.

a, Haematoxylin and eosin-stained bronchioles and blood vessels from the lungs of mice treated with vehicle (10% DMSO) and challenged with PBS or CRA, or treated with 30 mgkg⁻¹ 12,13-diHOME (solubilized in 10% DMSO) and challenged with CRA. Scale bars, 50 μm. Representative of four experimental repeats. b, Peritoneal 12,13-diHOME treatment (n = 22 mice) increases serum IgE compared with vehicle-treated CRA-challenged mice (n = 19 mice; linear mixed effects (LME), P= 0.019) or vehicle-treated, PBS-challenged mice (n = 14 mice; LME, P= 0.0088). c,d, 12,13-diHOME treatment (n = 19 mice) increases the number of infiltrating cells surrounding the bronchioles (c) and veins (d) of CRA-challenged mice compared with mice treated with vehicle and challenged with PBS (n = 10 mice; LME, P = 0.0202 (bronchial), P = 0.000122 (venous)) or mice treated with vehicle and challenged with CRA (n = 18 mice; LME, P=0.0449 (bronchial), P = 0.00127 (venous)). e, Peritoneal 12,13-diHOME treatment increases the number of lung-resident T cells (CD3⁺) in CRA-challenged mice (n = 8 mice) compared with mice treated with vehicle and challenged with PBS (n = 10 mice; LME, P = 4.69×10⁻⁶) or mice treated with vehicle and challenged with CRA (n = 10 mice; LME, P = 0.016). f, 12,13-diHOME treatment of mice challenged with CRA (n = 8 mice) increases the expression of IL1B compared with expression in mice treated with vehicle and challenged with PBS (n = 9 mice; LME, P=0.0010) or mice treated with vehicle and challenged with CRA (n = 9 mice; LME, P = 0.030). g, 12,13-diHOME treatment of mice challenged with CRA (n = 18 mice) decreases the number of T_reg cells compared with the number in mice treated with vehicle and challenged with CRA (n = 18 mice; LME, P= 0.031). h, Peritoneal treatment of mice with 12,13-diHOME significantly increases the concentration of 12,13-diHOME in the lungs (n = 7 mice; LME, P = 0.0048) at 3h after delivery. Unique symbols (triangles, squares, diamonds, circles, pluses, asterisks and crosses) represent mice from independent repeats. All data were plotted using boxplots from ggplot2, which displays the median, the 25th and 75th percentiles, and the smallest and largest values within 1.5× the interquartile range (whiskers). See also Supplementary Fig. 1.

Fig. 2 |. 12,13-diHOME acts through PPARγ on DCs to decrease the number of Tregs.

a, Treatment with 12,13-diHOME causes a dose-dependent decrease in IL-10 secretion from human DCs (n = 3 human donors; LME, P = 3.67×10⁻³, P= 2.68 ×10⁻⁶ and P = 7.36×10⁻⁷ for concentrations of 75μM, 130μM and 200 μM, respectively). b, Treatment of human DCs with 130 μM 12,13-diHOME causes a specific decrease in the frequency of T_reg cells (n = 4 human donors; CD3⁺CD4⁺CD25⁺FOXP3⁺; LME, P= 0.00066). c, Treatment of human DCs with 130μM 12,13-diHOME causes changes in gene expression consistent with activation of PPARγ (n = 4 human donors; LME, P = 2.86×10⁻⁹, P= 5.88×10⁻⁸ and P = 1.42×10⁻⁹ for CD1a, FABP4 and HADH, respectively). Fold changes (2^−ΔΔC_t) relative to vehicle control are presented on a log scale. d-f, 12,13-diHOME decreased the expression of CD1a (d), CCR7 (e) and CD80 (f) on human DCs (CD3⁻CD19⁻CD11c⁺; n = 4 human donors; LME, comparing vehicle with 75 μM 12,13-diHOME the P values were P<2.0×10⁻¹⁶, P< 3.1 ×10⁻⁴ and P<3.6×10⁻¹² for CD1a, CCR7 and CD80, respectively). g, Raw264.7 cells transfected with a PPARγ-activated luciferase reporter and treated with 12,13-diHOME or known PPARγ agonists GW1929 (K_d = 1.4nM) and rosiglitazone (K_d = 40 nM) (n = 3 independent experiments; graph depicts median and s.e.m. for all datapoints). For a-f, data are presented as boxplots generated using ggplot2, which displays the median, the 25th and 75th percentiles, and the smallest and largest values within 1.5× the interquartile range (whiskers). For a-g, experiments were performed with n = 3–4 treatment replicates using cells isolated from three or four independent donors (biological replicates are represented by triangles, diamonds, circles and squares). See also Supplementary Fig. 2.

Fig. 3 |. Neonatal gut-microbiome-derived EH genes from B. bifidum and E. faecalis produce 12,13-diHOME and decrease the number of lung T_reg cells in mice challenged with CRA.

a, Neonates who develop childhood atopy and/or asthma exhibit increased faecal concentrations of 12,13-diHOME (n = 41 neonates; LR adjusting for race, P= 0.037; see Supplementary Table 6 for a full summary of the statistical analysis). b, Neonates who develop atopy and/ or asthma during childhood have more bacterial EH genes in their stool (n = 26 neonates; LR adjusting for formula feeding; P = 0.007; Supplementary Table 7). c, The 30 most abundant EH genes identified in stool are enriched in neonates who develop atopy. Dendograms represent hierarchical clustering of bacteria and genes generated using HClust2. NA, not applicable. d, Three of the candidate EH genes, E. faecalis NP_814872, B. bifidum YP_003971091 and B. bifidum YP_003971333, convert 12,13-EpOME to its conjugate diol, 12,13-diHOME. e, Mice orally gavaged with LA and E. coli overexpressing NP_814872, YP_003971091 and YP_003971333 (3EH and LA) have significantly more 12,13-diHOME in their plasma than those supplemented with vehicle (LME; no CRA n= 5 mice, P= 0.038; CRA-challenged n = 9 mice, P = 0.0065). f, Mice treated with 3EH and LA and challenged intratracheally with CRA had significantly fewer lung Teg cells than mice treated with vehicle and challenged with CRA (LME; CRA-challenged n= 9 mice, P= 6.57×10⁻⁵). For a, b, e and f, data are presented as boxplots that were generated using ggplot2, which displays the median, the 25th and 75th percentiles, and the smallest and largest values within 1.5× the interquartile range (whiskers). See also Supplementary Fig. 3 and Supplementary Tables 6 and 7.

Fig. 4 |. Increased concentrations of 12,13-diHOME and the 3EH genes in neonatal stool are associated with the development of childhood atopy, eczema and/or asthma in two US cohorts.

a, Increased bacterial 3EH copy number is observed in neonates who developed atopy and/or asthma, and corresponds with the significant increase in overall faecal EH genes identified by ShortBred analysis (n = 41 neonates; LR, P = 0.0387; Fig. 3b, Supplementary Table 10). Data are presented as a boxplot that was generated using ggplot2, which displays the median, the 25th and 75th percentiles, and the smallest and largest values within 1.5× the interquartile range (whiskers). b, In a subset of the WHEALS cohort, an increase in 12,13-diHOME concentration in neonatal stool significantly increased the relative probability of developing atopy at two years of age and/or asthma at four years of age (n = 41 neonates; LR, 3EH⁺: OR = 1.25, 95% CI = 0.98–1.59, P = 0.073; 12,13-diHOME adjusted for race: OR = 5.07, 95% CI =1.10–23.3, P = 0.037; see Supplementary Table 12 for a full summary of the statistical analysis). For 3EH, analysis was performed using a log₂ transformation of the 3EH copy number. c, In a subset of the TIPS birth cohort, an increase in faecal 3EH copy number was associated with a significantly increased relative probability of developing eczema in two-year-old children and/or asthma in four-year-old children (n = 50 neonates; LR, 3EH⁺: OR = 1.25, 95% CI = 1.05–1.50, P = 0.013; 12,13-diHOME: OR = 1.51, 95% CI = 0.95–2.40, P = 0.079; see Supplementary Table 14 for a full summary of the statistical analysis). For 3EH, analysis was performed using a log₂ transformation of the 3EH copy number. The OR and CI for each risk factor are shown in b and c. See also Supplementary Fig. 3 and Supplementary Tables 6–14.