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. 2021 Nov 1;218(11):e20210235.
doi: 10.1084/jem.20210235. Epub 2021 Oct 6.

Maternal gut microbiome regulates immunity to RSV infection in offspring

Wendy Fonseca  1 , Carrie-Anne Malinczak  1 , Kei Fujimura  2 , Danny Li  2 , Kathryn McCauley  2 , Jia Li  3 , Shannon K K Best  1 , Diana Zhu  1 , Andrew J Rasky  1 , Christine C Johnson  3 , Jennifer Bermick  4 , Edward M Zoratti  5 , Dennis Ownby  6 , Susan V Lynch  2 , Nicholas W Lukacs  1   7 , Catherine Ptaschinski  1   7
Affiliations

Maternal gut microbiome regulates immunity to RSV infection in offspring

Wendy Fonseca et al. J Exp Med. .

Abstract

Development of the immune system can be influenced by diverse extrinsic and intrinsic factors that influence the risk of disease. Severe early life respiratory syncytial virus (RSV) infection is associated with persistent immune alterations. Previously, our group had shown that adult mice orally supplemented with Lactobacillus johnsonii exhibited decreased airway immunopathology following RSV infection. Here, we demonstrate that offspring of mice supplemented with L. johnsonii exhibit reduced airway mucus and Th2 cell-mediated response to RSV infection. Maternal supplementation resulted in a consistent gut microbiome in mothers and their offspring. Importantly, supplemented maternal plasma and breastmilk, and offspring plasma, exhibited decreased inflammatory metabolites. Cross-fostering studies showed that prenatal Lactobacillus exposure led to decreased Th2 cytokines and lung inflammation following RSV infection, while postnatal Lactobacillus exposure diminished goblet cell hypertrophy and mucus production in the lung in response to airway infection. These studies demonstrate that Lactobacillus modulation of the maternal microbiome and associated metabolic reprogramming enhance airway protection against RSV in neonates.

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Conflict of interest statement

Disclosures: S.V. Lynch reported personal fees from Siolta Therapeutics Inc. during the conduct of the study and outside the submitted work; in addition, S.V. Lynch had a patent to Reductive prodrug cancer chemothera (Stan449-PRV) issued, a patent to combination antibiotic and antibody therapy for the treatment of Pseudomonas aeruginosa infection (WO 2010091189 A1) issued, a patent to therapeutic microbial consortium for induction of immune tolerance with royalties paid to Siolta Therapeutics Inc., a patent to systems and methods for detecting antibiotic resistance (WO 2012027302 A3) issued, a patent to nitroreductase enzymes (US 7687474 B2) issued, a patent to sinusitis diagnostics and treatments (WO 2013155370 A1) issued, a patent to methods and systems for phylogenetic analysis (US 20120264637 A1) issued, a patent to methods and compositions relating to epoxide hydrolase genes licensed to Siolta Therapeutics Inc., and a patent to novel Lactobacillus and Micrococcus species that promote tolerogenic immunity pending. No other disclosures were reported.

Figures

Figure 1.
Figure 1.
Maternal supplementation with L. johnsonii regulates offspring immunity to RSV. (A) Experimental design. Female BALB/c mice were supplemented with L. johnsonii daily for 7 d before mating, then twice weekly until delivery. Neonates were infected with RSV at 7 d old, and samples were collected on days 12–14. (B) Histology sections of infected neonates were stained with H&E to visualize inflammation and PAS to visualize mucus production. (C) Expression of the mucus-associated gene gob5 was measured by qPCR. Fold changes was measured compared with uninfected offspring. (D) Expression of the gene for the RSV F protein was measured by qPCR. (E) Cytokine production from mediastinal lymph node cells following RSV restimulation was measured by Bioplex assay. (F–I) The following cells populations were measured in the lungs of control and RSV-infected neonates at 7 d after infection by flow cytometry: (F) ILC2s, (G) eosinophils, (H) CD4+ T cells, and (I) regulatory T cells. Data represent the mean ± SE from four to eight mice (experimental repeats three or four). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure S1.
Figure S1.
L. johnsonii–dependent protection from RSV-induced pathology persists into maturity. (A) Experimental design. Female mice were supplemented with L. johnsonii for 7 d before mating, then twice weekly until delivery, while control mice were treated with PBS. Offspring were weaned, then infected with RSV at 6 wk of age. Samples were taken at 8 d after infection. (B) Histology sections of the lungs were stained with H&E to visualize inflammation or PAS to visualize mucus. (C) Expression of the mucus-associated gene gob5 was measured in lung tissue by qPCR. (D) Mediastinal lymph nodes were digested into a single-cell suspension and restimulated with RSV. Cytokine production was measured by Bioplex assay. (E–G) The following cell populations in the lung were measured by flow cytometry: (E) CD4+ T cells, (F) CD11c+CD11b+ DCs, and (G) CD11c+CD103+ DCs. Data represent the mean ± SE from four or five mice from two experimental repeats. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.
Figure 2.
Figure 2.
Prenatal supplementation of L. johnsonii altered gut microbiome of mothers and offspring. (A) Bacterial β–diversity between the two groups of mothers, presenting different bacterial β–diversity as shown in the principal coordinate analysis (PERMANOVA; R2 = 0.177; P = 0.002; Unweighted UniFrac). (B) Differentially enriched taxa between mothers receiving L. johnsonii supplementation and mothers receiving PBS supplementation. The size of the circle represents the normalized mean of a given sequence variant in a group of mothers. (C) Principal coordinate (PC) analysis of cecal microbiome of offspring that were either uninfected or infected with RSV, presenting different bacterial β–diversity (PERMANOVA; R2 = 0.341; P = 0.01; Unweighted UniFrac). (D) Differentially enriched taxa between offspring of L. johnsonii–supplemented mothers and offspring of PBS-supplemented mothers. The size of the circle represents the normalized mean of a given sequence variant in a group of offspring. (E) Unweighted UniFrac distance comparisons between groups of offspring from control or supplemented females, and control or RSV-infected. Each bar represents the mean distance between mice within a given treatment. Each experiment analyzed four samples per group.
Figure S2.
Figure S2.
Metabolic profiles of mothers and offspring plasma and neonates ingested milk. Heatmap of differentially expressed metabolites. The row displays metabolites that achieved statistical significance (P ≤ 0.05), and fold change between LJ and PBS groups is presented. Metabolites significantly decreased were displayed in green, while metabolites significantly increased were displayed in red. The brightness of each color corresponded to the magnitude of the difference when compared with average value. (A) Differentially expressed metabolites in the plasma of supplemented females. (B) Differentially expressed metabolites in the plasma of offspring. (C) Differentially expressed metabolites in the milk of supplemented females. Each experiment analyzed four samples per group.
Figure 3.
Figure 3.
Postnatal regulation of immunity to RSV by maternal supplementation of L. johnsonii. (A) Experimental design. Female BALB/c mice were supplemented with L. johnsonii daily for 7 d before mating, then twice weekly until delivery. Cross-fostered offspring from PBS supplemented mothers to a L. johnsonii–supplemented mother (PBS-LJ) and vice versa (LJ-PBS). As controls, PBS-supplemented mothers and L. johnsonii–supplemented mothers between the same group were swapped to obtain PBS-PBS and LJ-LJ groups, offspring were infected with RSV at 7 d old, and samples were collected on days 12–14. (B) Histology sections of infected neonates were stained with PAS to visualize mucus production. (C) Expression of the mucus-associated gene gob5 was measured by qPCR. (D) Expression of the gene muc5ac was measured by qPCR. Fold changes were measured compared with uninfected offspring. (E) Expression of the gene for the RSV F protein was measured by qPCR. Data represent the mean ± SE from four to seven mice (experimental repeats three or four). *, P ≤ 0.05; **, P ≤ 0.01.
Figure 4.
Figure 4.
Prenatal regulation of adapted immunity to RSV by maternal L. johnsonii supplementation. (A–C) LDLN cells were restimulated with RSV, and production of IL-4, IL-5, and IL-13 in culture supernatant was measured by Bioplex. (D–J) Total numbers of (D) ILC2s: Lin-CD45+CD90+ST2+c-Kit+CD127+GATA3+; (E) eosinophils: SSChighCD11b+SiglecF+; (F) interstitial macrophages: CD11b+CD11c−F4/80+; (G) DCs: CD11b+CD11c+MHCII+ and CD11c+MHCII+CD11b−CD103+; (H) regulatory T cells: CD3+CD4+Foxp3+; (I) CD4+ T cells: CD3+CD4+, and activated CD3+CD4+CD69+; and (J) CD8+ T cells: CD3+CD8+ and activated CD3+CD8+CD69+. Data analysis was performed using FlowJo software. Data represent the mean ± SE from four to eight mice per group from three or four experimental repeats. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
Figure 5.
Figure 5.
Maternal supplementation with L. johnsonii alters offspring gut microbiome. (A) Principal coordinate (PC) analysis of cecal microbiome of only cross-fostered offspring that were challenged with RSV (PERMANOVA; R2 = 0.413; P = 0.001; Unweighted UniFrac). (B) Unweighted UniFrac distance comparisons between groups of offspring challenged with RSV. Each bar represents the mean distance between mice within a given treatment (prenatal and postnatal supplementation outcomes). (C) Differentially enriched taxa between mice receiving prenatal and postnatal PBS supplementation and mice receiving prenatal and postnatal L. johnsonii supplementation. The size of the circle represents the normalized mean of a given sequence variant in a group of offspring.
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