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. 2023 Mar 10:4:1135412.
doi: 10.3389/falgy.2023.1135412. eCollection 2023.

Dysbiotic lung microbial communities of neonates from allergic mothers confer neonate responsiveness to suboptimal allergen

Jeffery C Bloodworth  1   2 , Aki Hoji  1 , Garen Wolff  1   3 , Rabindra K Mandal  1 , Nathan W Schmidt  1   2 , Jessy S Deshane  4 , Casey D Morrow  5 , Kirsten M Kloepfer  3 , Joan M Cook-Mills  1   2
Affiliations

Dysbiotic lung microbial communities of neonates from allergic mothers confer neonate responsiveness to suboptimal allergen

Jeffery C Bloodworth et al. Front Allergy. .

Abstract

In humans and animals, offspring of allergic mothers have increased responsiveness to allergens. This is blocked in mice by maternal supplementation with α-tocopherol (αT). Also, adults and children with allergic asthma have airway microbiome dysbiosis with increased Proteobacteria and may have decreased Bacteroidota. It is not known whether αT alters neonate development of lung microbiome dysbiosis or whether neonate lung dysbiosis modifies development of allergy. To address this, the bronchoalveolar lavage was analyzed by 16S rRNA gene analysis (bacterial microbiome) from pups of allergic and non-allergic mothers with a basal diet or αT-supplemented diet. Before and after allergen challenge, pups of allergic mothers had dysbiosis in lung microbial composition with increased Proteobacteria and decreased Bacteroidota and this was blocked by αT supplementation. We determined whether intratracheal transfer of pup lung dysbiotic microbial communities modifies the development of allergy in recipient pups early in life. Interestingly, transfer of dysbiotic lung microbial communities from neonates of allergic mothers to neonates of non-allergic mothers was sufficient to confer responsiveness to allergen in the recipient pups. In contrast, neonates of allergic mothers were not protected from development of allergy by transfer of donor lung microbial communities from either neonates of non-allergic mothers or neonates of αT-supplemented allergic mothers. These data suggest that the dysbiotic lung microbiota is dominant and sufficient for enhanced neonate responsiveness to allergen. Importantly, infants within the INHANCE cohort with an anti-inflammatory profile of tocopherol isoforms had an altered microbiome composition compared to infants with a pro-inflammatory profile of tocopherol isoforms. These data may inform design of future studies for approaches in the prevention or intervention in asthma and allergic disease early in life.

Keywords: allergy; lung; microbiome; neonates; tocopherol.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Enhanced responsiveness to challenge with HDM or OVA by pups of mothers with allergy was inhibited by maternal supplementation with α-tocopherol. The allergen of the mother and offspring can differ. (A,C) Allergic and non-allergic mothers received basal diet or diet supplemented with αT (250 mg αT/kg of diet) during pregnancy and nursing. Timeline for allergen-sensitization and allergen-challenge of mothers and offspring. (B,D) Pup BAL eosinophils, monocytes, lymphocytes and neutrophils. (E) Relative IL-5 mRNA expression in lungs of HDM-challenged pups of allergic and non-allergic mothers with basal or αT-supplemented diets. BAL, bronchoalveolar lavage. n = 8–10 mice/group. Saline treated pups did not have allergic inflammation (data not shown). *p < 0.05.
Figure 2
Figure 2
Pups of allergic mothers have altered lung bacteria microbial composition. Mouse treatments were as in (Figure 1A). BAL microbiota from pups at (A) PND4 and (B,C) 24 h after OVA-challenge (PND16) was separated and analyzed by 16S rRNA gene sequencing and a microbiome analysis pipeline. (A) At PND4, before allergen exposure, there was increased Proteobacteria and decreased Bacteroidota in the lungs of offspring of allergic mothers (log2FC > 0.6, FDR < 0.1) as compared to offspring of non-allergic mothers with basal diet. (B) ASV table of the relative abundance of phyla within the total pup BAL bacterial microbiome. *p < 0.05 compared to other groups. (C) BAL microbiota from pup BAL PND16 were separated and concentrated by differential centrifugation as in the methods, suspended in minimal volume for fixation in a small spot on a glass slide and stained with gram stain from bacteria. Representative images of lung microbiota are shown.
Figure 3
Figure 3
Pups of allergic mothers have altered bacteria microbiome. Mouse treatments were as in (Figure 1A). Pup BAL microbiota were separated and analyzed by 16S rRNA gene sequencing at PND16. Shown are the % abundance for pup BAL bacteria with a significant difference in the OVA/Basal group compared to the other groups. *p < 0.05.
Figure 4
Figure 4
After intranasal microbiome transfers and airway allergen challenge, there was pup BAL microbiota with increased Proteobacteria and decreased Bacteroidota taxa for pups that were either recipient pups of mothers in the OVA,basal group or were pups receiving microbiome from pups of mothers in the OVA,basal group. (A) Timeline for treatment of mothers and pups. (B) Donor BAL microbiome was administered intranasally in 10 µl to PND4 recipient pups (as indicated in figures as the group of pups providing donor BAL microbiome for transfer into a recipient group of pups, i.e., donor → recipient group). Yellow arrows on the x-axis are those groups with donor and recipients within the same group. In RED BOX are groups with recipient or donor microbiota of PND16 pups of allergic mothers (OVA/basal). Blue arrows within panel B indicate that Bacteroidota are decreased and Gamma-Proteobacteria are increased in groups in red box. N = 8/group. In panels B,C only, the OVA was in 0.09% saline; nevertheless, it did not alter the fold effect on BAL cell inflammation which is included in (Figure 5) with data from 7 microbiome transfer experiments. (C) In RED BOX are recipient or donor microbiota of PND16 pups from allergic mothers (OVA/basal). Data are presented as percent abundance of bacteria taxa. *, p < 0.05 as compared to Saline,basal → Saline,basal group (yellow arrow in graphs in C). Sal/B, saline-treated mother with basal diet. Sal/αT, saline-treated mother with αT-supplemented diet. OVA/B, OVA allergen-treated mother with a basal diet. OVA/αT, OVA allergen-treated mother with αT-supplemented diet.
Figure 5
Figure 5
Recipient or donor microbiota from pups of allergic mothers (OVA,basal) conferred responsiveness to allergen in the recipient pups (red box). Mice were treated as in timeline in (Figure 4A) BAL (A) eosinophils, (B) monocytes, (C) lymphocytes, and (D) neutrophils are presented as mean ± SEM. Data are from 7 experiments. N = 10–36/group. Sal/B, saline-treated mother with basal diet. Sal/αT, saline-treated mother with αT-supplemented diet. OVA/B, OVA allergen-treated mother with a basal diet. OVA/αT, OVA allergen-treated mother with αT-supplemented diet. *p < 0.05 as compared to the saline,basal → saline,basal group., +p < 0.1 as compared to no donor → Saline/basal group.
Figure 6
Figure 6
Recipient or donor microbiota from pups of allergic mothers (OVA,basal) conferred allergen sensitization with increased IgE but not increased IgG2b or IgG1 (red box). Mice were treated as in timeline in (Figure 4A). Serum (A) anti-OVA IgE, (B) anti-OVA IgG2b, and (C) anti-OVA IgG1 as determined by ELISA. Data are presented as mean ± SEM. N = 6–9/group. Sal/B, saline-treated mother with basal diet. Sal/αT, saline-treated mother with αT-supplemented diet. OVA/B, OVA allergen-treated mother with a basal diet. OVA/αT, OVA allergen-treated mother with αT-supplemented diet. *p < 0.05 as compared to the saline,basal → saline,basal group. +p < 0.1 as compared to no donor → Saline/basal group.
Figure 7
Figure 7
Recipient or donor microbiome from pups of allergic mothers (OVA,basal) conferred allergen-induced increases in CCL11, IL-13, IL-5, IL-33, and Muc5ac(red box). Mice were treated as in timeline in (Figure 4A). Lung cytokine expression was determined by qPCR. (A) CCL11. (B) IL-13. (C) IL-5. (D) IL-33. (E) Muc5ac. N = 6–9/group. Data are presented as mean ± SEM. N = 6–9/group. Sal/B, saline-treated mother with basal diet. Sal/αT, saline-treated mother with αT-supplemented diet. OVA/B, OVA allergen-treated mother with a basal diet. OVA/αT, OVA allergen-treated mother with αT-supplemented diet. *p < 0.05 as compared to the saline,basal → saline,basal group. **p < 0.05 as compared to saline/αT → saline/αT group.
Figure 8
Figure 8
Infants with an anti-inflammatory tocopherol isoform profile (higher αT, lower γT) for allergic responses had a different abundance of bacterial microbiota compared to other tocopherol isoform profiles. Serum αT and γT for infants in the INHANCE cohort were measured by HPLC. Four groups of infants (Q1, Q2, Q3, Q4) for 3–5 months and for 12–18 months infants were generated using high and low αT and γT concentrations in (Table 1) that were defined as higher or lower than the median concentration for the tocopherol isoform for the age group. (A) 3–5 months and (B) 12–18 months of life. p values are given for significant differences or trends in taxa compared to group Q4.
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