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. 2020 Sep 11;15(9):e0238923. doi: 10.1371/journal.pone.0238923

Maternal supplementation with Bifidobacterium breve M-16V prevents their offspring from allergic airway inflammation accelerated by the prenatal exposure to an air pollutant aerosol

Chiyoko Terada-Ikeda 1,#, Masahiro Kitabatake 1,#, Akari Hiraku 2, Kumiko Kato 3, Satsuki Yasui 1, Natsuko Imakita 1, Noriko Ouji-Sageshima 1, Noriyuki Iwabuchi 2, Kaoru Hamada 4, Toshihiro Ito 1,*
Editor: Heinz Fehrenbach5
PMCID: PMC7485856  PMID: 32915886

Abstract

Bifidobacterium breve M-16V is a probiotic bacterial strain with efficacy in infants achieved by suppressing T-helper type (Th) 2 immune responses and modulating the systemic Th1/Th2 balance. Exposure to air pollution during pregnancy increases asthma susceptibility in offspring. The aim of this study was to investigate the effects of the maternal intake of B. breve M-16V on susceptibility to asthma accelerated by prenatal exposure to air pollution. The intake of B. breve M-16V in residual oil fly ash (ROFA)-exposed pregnant mice resulted in fewer eosinophils in the bronchoalveolar lavage fluid of neonatal mice and reduced allergic lung inflammation. The expressions of Th2 cytokines including IL-5 and IL-13 were decreased in neonatal mice from ROFA-exposed mothers fed B. breve M-16V. The analysis of fecal microbiota from neonatal mice revealed that the intake of B. breve M-16V by mothers changed the composition of fecal microbiota in neonatal mice, which resulted in a decreased population of Firmicutes. Moreover, several bacterial strains of fecal microbiota from neonatal mice had a strong correlation with Th2 cytokines and histological score. These results suggest that the maternal intake of M-16V might have beneficial effects in neonates by preventing and/or alleviating allergic reactions accelerated by prenatal exposure to air pollution.

Introduction

The increasing incidence of asthma in early life suggests that prenatal environmental exposure may influence susceptibility to allergic airway diseases [1–3]. Indeed, recent studies suggested that prenatal exposure to air pollution enhanced the risk of developing asthma in offspring [4–6]. Epidemiological and experimental studies reported that diesel exhaust particles (DEP), a major component of particulate air pollution in many urban areas, stimulated sensitization to allergens [7, 8]. In addition, we and other groups found that a surrogate for ambient air pollution, residual oil fly ash (ROFA) from an oil combustion power plant, contributed to increased asthma susceptibility in offspring [9–11].

The pathogenesis of asthma is characterized by a large influx of eosinophils and CD4+ T-helper (Th) cells, especially Th2 cells, around lung airways [12]. Th2 cells synthesize high levels of IL-4, IL-5, and IL-13, which induce immunoglobulin (Ig) E synthesis and eosinophilic inflammation [13].

Recent studies on the properties and functionality of living micro-organisms in food have suggested that probiotics play an important role in immunological development, as well as digestive and respiratory functions [14, 15]. Numerous animal and human studies reported that the intake of probiotics such as lactobacilli and bifidobacteria inhibited allergic responses and allergic sensitization by inhibiting Th2 responses [16–18]. There are many species within the genus Bifidobacterium, among which Bifidobacterium breve is one of the most abundant in human neonates [19].

B. breve M-16V is a probiotic strain isolated from the fecal sample of a human baby [20]. M-16V has been used for low-birth-weight infants in Australia and Japan [21–23]. It was previously shown that the administration of M-16V to infants with food allergy and atopic dermatitis significantly improved their allergic symptoms [15, 24, 25]. M-16V administration was associated with a significant increase in the proportion of Bifidobacteria and a decrease in the proportion of aerobic bacteria in the fecal microbiota [15, 24]. In addition, Inoue et al. demonstrated that M-16V modulated the Th1/Th2 balance in OVA-immunized mice, which are commonly used as a Th2-induced bronchial asthma model [16]. However, whether M-16V affects asthma susceptibility in offspring induced by air pollution is unknown, and a detailed correlation between fecal microbiota and Th2 responses in lungs requires further study. We previously established a mouse model to investigate the potential effects of air pollution exposure during pregnancy on the susceptibility of offspring to asthma [11].

In the present study, we evaluated the effect of M-16V on the pathogenesis of allergic asthma in our experimental asthma model with prenatal exposure to air pollutants using ROFA, and analyzed bacterial organisms in the fecal microbiota that correlated with asthma responses.

Materials and methods

M-16V preparation

B. breve M-16V powder was supplied by the Morinaga Milk Industry Co., Ltd. (Kanagawa, Japan). Pure cells were freeze-dried with dextrin to achieve the desired concentration.

Animal studies

Animals

Pregnant BALB/c mice obtained from Japan SLC (Hamamatsu, Japan) were maintained at 25°C with a 12-h light/dark cycle in an animal facility of the Department of Animal Resources at Nara Medical University. Mice in the control group were fed an AIN-93G-modified basal diet (Oriental Bio Service Inc., Kyoto, Japan) and mice in the M-16V group were fed a basal diet supplemented with M-16V. The M-16V group was fed approximately 0.5 g of the AIN-93G diet with the addition of 1 million viable cells of M-16V. The control group was fed 0.5 g of the AIN-93G diet with the addition of dextrin. Each group was fed separately from their usual diet. Mothers were fed during the examination period with AIN-93G (Control) or M-16V once a day. The diet of the mothers was continued throughout the nursing time until the analysis of the offspring. Each mother was housed with her offspring in a single cage and all neonates (both male and female) were breastfed throughout the study (Fig 1). The Animal Care and Use Committee at Nara Medical University approved all animal experiments conducted in this study (approval No.11367), and all methods were carried out based on the Policy on the Care and Use of Laboratory Animals, Nara Medical University. These experiments were carried out from 2015 to 2018.

Fig 1. Schematic representation of the experimental protocol.

Fig 1

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(A) Analysis of OVA allergic neonates from mothers exposed to PBS (no ROFA) on a control (AIN-93G) or M-16V diet. On days 5, 3, and 1 before delivery, normal pregnant BALB/c female mice were exposed to aerosolized PBS. The newborns received a single intraperitoneal injection of OVA (5 μg) and alum (0.5 mg) at day 3, followed by exposure to aerosolized 1% OVA on days 26–28 of life. (B) Analysis of OVA allergic neonates from PBS- or ROFA-exposed mothers on a control (AIN-93G) diet. On days 5, 3, and 1 before delivery, normal pregnant BALB/c female mice were exposed to aerosolized ROFA leachate (50 mg/ml) or PBS. The newborns received a single intraperitoneal injection of OVA (5 μg) and alum (0.5 mg) at day 3, followed by exposure to aerosolized 1% OVA on days 26–28 of life. (C) Analysis of OVA allergic neonates from mothers exposed to ROFA on a control (AIN-93G) or M-16V diet. On days 5, 3, and 1 before delivery, normal pregnant BALB/c female mice were exposed to aerosolized ROFA leachate (50 mg/ml). The newborns received a single intraperitoneal injection of OVA (5 μg) and alum (0.5 mg) at day 3, followed by exposure to aerosolized 1% OVA or PBS on days 26–28 of life. All Analyses in this study were performed 48 h after the final aerosol exposure.

Study protocol

Fig 1 summarizes the experimental protocols used in this study. ROFA obtained from the precipitator unit of a local power plant was kindly provided by Dr. Lester Kobzik (Harvard School of Public Health, Boston) [11]. ROFA was suspended in phosphate buffered saline (PBS) to 100 mg/ml and sonicated for 10 min. The ROFA suspension was incubated at 37°C with rotation for 4 h and then centrifuged at 3000 ×g for 10 min. The supernatant (leachate) was removed and diluted to 50 mg/ml in PBS. As described previously [11], pregnant BALB/c mice were exposed to nebulized PBS or ROFA leachate for 30 min at days 14, 16, and 18 of pregnancy. After birth, neonatal mice were intraperitoneally sensitized to 5 μg of ovalbumin (OVA; Grade III) (Sigma Chemical, St. Louis, MO) and 0.5 mg of alum (Nacalai Tesque Inc., Kyoto, Japan) diluted in 50 μl PBS. On days 26–28 of life, neonatal mice were exposed to aerosolized OVA (1%, 10 min/day, for 3 consecutive days) or PBS.

Pathologic analysis

At 48 h after the final challenge, neonatal mice were euthanized by blood collection from the left ventricle of the heart following pentobarbital (Nacalai Tesque Inc.) anesthesia. The trachea was cannulated and 0.5 ml of sterile PBS was instilled to harvest the bronchoalveolar lavage fluid (BALF), which was centrifuged at 800 ×g for 5 min, and resuspended in 0.5 ml of PBS. Differential cell counts were performed on cytocentrifuge slides prepared by centrifugation of samples at 800 rpm for 5 min using Cytospin 3 (Thermo Fisher Scientific Inc., Waltham, MA). The slides were fixed in 95% methanol and stained with Diff-Quik (Sysmex, Kobe, Japan), modified Wright-Giemsa stain, and then 200 cells were counted for each sample by microscopy. Macrophages, lymphocytes, neutrophils, and eosinophils were enumerated. BALF supernatants were analyzed for the measurement of cytokines (IL-4, IL-5, IL-13) using mouse ELISA kits according to the manufacturer’s protocol (Thermo Fisher Scientific Inc.). After lavage, the left lobe of the lung was removed, inflated, and fixed with 4% paraformaldehyde for histological assessment. After paraffin embedding, sections for microscopy were stained with Periodic acid-Schiff (PAS) to visualize mucus production. Sections were scored based on the mucus production of the bronchi as follows: 1 –minimal (<25%), 2 –slight (<25%–50%), 3 –moderate (<50%–75%), 4 –severe (>75%), as previously described [11, 26]. The mean score from at least 10 bronchi per section was assessed as the mucin score.

Quantitative real-time PCR (qPCR)

The right lobe of the lung was immersed in 0.2 ml RNAlater® Stabilization Solution (Thermo Fisher Scientific Inc.) overnight at 4°C and stored at −80°C until RNA extraction. Total RNA was isolated using NucleoSpin® RNA (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and stored at −80°C. Total RNA was extracted and 1 μg of total RNA was reverse-transcribed to cDNA according to the procedure previously described [27]. qPCR was performed with TaqMan gene expression assays using a Step One™ qPCR system (Thermo Fisher Scientific Inc.). TaqMan gene expression assays for Gapdh (Mm99999915), Il4 (Mm00445259), Il5 (Mm00439646), Il13 (Mm00434204), and Muc5ac (Mm0126718) were purchased from Thermo Fisher Scientific Inc. mRNA expression was analyzed by the ΔΔCt method and normalized to GAPDH expression as previously described [27].

Microbiota profiling

DNA extraction from fecal samples

Fecal samples were collected at 48 h after final challenge and stored at −80°C until analysis. DNA was extracted using the bead-beating method as previously described [28] with some modifications. Fecal samples (20 mg) were suspended in 450 μl extraction buffer (100 mM Tris/HCl, 40 mM EDTA, pH 9.0) and 50 μl 10% SDS. Glass beads (300 mg, 0.1 mm diameter) and 500 μl buffer-saturated phenol were added to the suspension and the mixture was vortexed vigorously for 180 s using a Multi-Beads Shocker (Yasui Kikai Co., Osaka, Japan) at a speed of 2,700 rpm. After centrifugation at 14,000 ×g for 5 min, 400 μl of the supernatant was extracted with phenol-chloroform and 250 μl of the supernatant was precipitated with isopropanol. Purified DNA was suspended in 2,000 μl of Tris-EDTA buffer (pH 8.0).

16S rRNA gene sequencing

Subsequent DNA extraction and sequencing by Illumina Miseq (Illumina Inc., San Diego, CA) were performed as described previously [29, 30]. Briefly, the V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using the Takara Ex Taq HS Kit (Takara Bio, Shiga, Japan) and the primer sets Tru357F (5′-CGCTCTTCCGATCTCTGTACGGRAGGCAGCAG-3′) and Tru806R (5′-CGCTCTTCCGATCTGACG-GACTACHVGGGTWTCTAAT-3′) with the following protocol: preheating at 94°C for 3 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and terminal extension at 72°C for 5 min. Then, 1 μl sample of the PCR product was amplified using the following barcoded primers adapted for Illumina MiSeq: Fwd 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3′ and Rev 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′, where X represents a barcode base. Amplification was performed according to the protocol described above except only eight cycles were performed. The products were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols. The purified products were quantified by Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). Equal amounts of amplicons were pooled and purified with the GeneRead Size Selection Kit (Qiagen) according to the manufacturer’s protocol. The pooled libraries were sequenced using an Illumina MiSeq instrument and the MiSeq v3 Reagent Kit (Illumina Inc.).

Sequencing data analysis

Briefly, phiX reads were removed from the raw Illumina paired-end reads and the sequences were analyzed using the QIIME software package version 1.8.0 (http://qiime.org/) [31, 32]. After removing potential chimeric sequences using UCHIME, assignment to operational taxonomic units (OTUs) using open-reference OTU picking [33] with a 97% threshold of pairwise identity was conducted. Subsequently, the taxonomical classification was performed using the Greengenes reference database (http://greengenes.secondgenome.com/downloads/database/13_5) [34]. UniFrac distances were calculated using QIIME version 1.8.0 software [31]. DNA sequences corresponding to the 16S rRNA gene data have been deposited in the DDBJ under accession number DRA010221.

Statistical analysis

Statistical significances in the gene expression, mucin score, and BAL analysis data were evaluated by analysis of variance. P values < 0.05 were considered to indicate statistically significant differences. Statistical analyses except for the analyses of gut microbiota were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Data are presented as the mean ± SEM and are representative of at least two independent experiments. The analyses of gut microbiota without the calculation of a false discovery rate (FDR) were performed using SPSS version 23.0 statistical software (IBM Corp., Armonk, NY). Intergroup differences were analyzed using the Mann-Whitney U-test and correlation analysis between gut bacteria and Th2 responses in the lungs were performed by Spearman’s correlation coefficient. Results were adjusted by FDR using the Benjamini and Hochberg method in R software version 3.6.0 (R Foundation for Statistical Computing, http://www.R-project.org/).

Results

ROFA during pregnancy accelerates allergic airway inflammation

We first compared the response of neonatal mice in the control (AIN-93G) and M-16V diet groups to OVA without ROFA exposure (Fig 1A). To examine the response in lungs, a histological assessment of lung tissue from mice euthanized at 48 h after the last challenge was conducted. The mice showed robust pathologic changes caused by allergic inflammation including an accumulation of eosinophils and mononuclear cells around the airways and vessels, and goblet cell hyperplasia. As shown in S1 Fig, there were no significant differences in mucin score or allergic Th2 gene expressions including IL-4, IL-5, IL-13, and Muc5ac between control (AIN-93G) and M-16V diet mice not exposed to ROFA. We previously reported offspring from mothers exposed to ROFA during pregnancy developed robust pathologic changes related to airway inflammation including mucin score [11]; therefore, we next assessed whether ROFA during pregnancy aggravated the response of neonatal mice to OVA in our model (Fig 1B). We confirmed that ROFA exposure of mothers on the control diet aggravated the response of their neonatal mice as assessed by mucin score and gene expressions of IL-5 and IL-13 (S2 Fig). Only ROFA exposed mice were used later and this was the focus in this study (Fig 1C).

B. breve M-16V attenuates lung allergic inflammation in offspring from ROFA-exposed mothers

Next, we examined whether B. breve M-16V prevented allergic inflammation accelerated by ROFA. Histological evaluation of neonatal OVA-exposed lungs from mothers exposed to ROFA during pregnancy revealed a reduction of pulmonary inflammation in the M-16V diet group compared with the control diet group (Fig 2A). Furthermore, mucus levels in the airways demonstrated a significant decrease in overall mucus production in the M-16V diet group (Fig 2B).

Fig 2. Histopathologic analysis of lung tissue.

Fig 2

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(A) Lung sections were stained with PAS to visualize mucus production at day 30 of life. (B) Quantitative analysis of mucus production using a scoring system of 1–4 detailed in the Materials and methods. The values are presented as the means ± SEM (n = 6–21). *P < 0.05 compared with OVA-sensitized and -exposed mice with a control diet.

Expressions of allergic response genes are decreased in OVA-allergic M-16V diet mice

To elucidate the mechanism underlying the changes in pulmonary inflammation between the control and M-16V diets, we evaluated the profile of inflammatory genes in the lungs. Gene expression levels of IL-5, IL-13, and Muc5ac were significantly lower in the OVA-allergic M-16V diet group compared with the control group (Fig 3B–3D). In addition, IL-4 gene expression tended to be decreased in the OVA-allergic M-16V diet group, although there was no statistically significant difference in IL-4 level between the groups (Fig 3A).

Fig 3. Effect of M-16V feeding on the expression of bronchial asthma-related genes.

Fig 3

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mRNA levels of IL-4 (A), IL-5 (B), IL-13 (C), and Muc5ac (D) in lungs were determined by real-time PCR. The values are presented as the means ± SEM (n = 6–21). *P < 0.05, **P < 0.01, compared with OVA-sensitized and -exposed mice with a control diet.

B. breve M-16V reduces eosinophil influx into airways

We also investigated the numbers of macrophages, lymphocytes, neutrophils, and eosinophils in the BALF. The number of eosinophils in the BALF was significantly decreased in the OVA-allergic M-16V diet group compared with the OVA-allergic control diet group, and there were no significant differences in the numbers of macrophages, neutrophils, and lymphocytes in OVA-exposed mice between the control and M-16V diet groups (Fig 4). Additionally, we measured cytokine production from the BALF. Although the gene expressions of IL-5 and IL-13 in the lungs were significantly lower in the OVA-allergic M-16V diet group compared with controls, no significant differences were found in IL-4, IL-5, or IL-13 protein production from the BALF (S3 Fig).

Fig 4. Effect of M-16V diet on the number of immune cells (macrophages, lymphocytes, neutrophils, and eosinophils) in the BALF.

Fig 4

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BALF was collected 48 h after the final aerosol challenge. The values are presented as the means ± SEM (n = 6–21). *P < 0.05 compared with OVA-sensitized and -exposed mice with a control diet.

B. breve M-16V diet changes the fecal microbiota profile

The M-16V diet prevented OVA-induced allergic inflammation in the lungs, and therefore we assessed the effect of M-16V on the fecal microbiota in neonatal mice. We compared the fecal microbiota composition of samples from neonatal mice with or without M-16V diet between control and OVA-allergic neonatal mice. The proportion of Actinobacteria was higher in the OVA-allergic M-16V diet group compared with the OVA-allergic control diet group, although there was no statistically significant difference (Fig 5A). No significant difference in the proportions of Bacteroides and Proteobacteria was observed in fecal samples between the control and B. breve M-16V diet groups (Fig 5B and 5C). However, the portion of Firmicutes was significantly lower in the B. breve M-16V diet group compared with the control group (Fig 5D).

Fig 5. Proportion of total bacteria species in feces between control and B. breve M-16V diet groups.

Fig 5

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The abundances of (A) Actinobacteria, (B) Bacteroides, (C) Proteobacteria, and (D) Firmicutes in fecal samples from each mouse were analyzed by 16S rRNA gene sequencing. The values are presented as the means ± SEM (n = 6–21). *P < 0.05 compared between control and B. breve M-16V diet OVA-allergic model mice.

Specific microbial taxa in feces are associated with allergy-related factors in lungs

Next, we identified which types of fecal microbiota were regulated by the B. breve M-16V diet and were associated with the pathogenesis of lung allergic responses in OVA-allergic mice. Using next-generation sequencing, several genera were associated with bronchial asthma-related factors such as lung histology and Th2 cytokines. In detail, Gemella and Streptococcus genera were significantly increased by the intake of B. breve M-16V in OVA-allergic mice, and these genera were negatively correlated with histological mucin score (Fig 6A–6D). In addition, the gene expression of the Th2 cytokine IL-5 was significantly correlated with the genera of Lactobacillus and Dehalobacterium. The B. Breve M-16V diet decreased the proportion of Lactobacillus and this had a positive correlation with IL-5 gene expression in the lungs. However, the B. Breve M-16V diet increased the proportion of Dehalobacterium and this had a negative correlation with IL-5 gene expression in the lungs (Fig 7A–7D). Moreover, gene expression of the Th2 cytokine IL-13 was also significantly correlated with two genera, Blautia and Faecalibacterium. While the B. Breve M-16V diet did not induce a significant proportion of Blautia or Faecalibacterium, both genera showed a negative correlation with IL-13 gene expression in the lungs (Fig 8A–8D). IL-5 expression level was not correlated with Blautia species and the IL-13 expression level was not correlated with Lactobacillus species. No microbiota genus was correlated with the gene expressions of IL-4 or Muc5ac.

Fig 6. Relationship of mucin score in lung histology and fecal microbiota composition of each sample.

Fig 6

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Relative abundance of (A) g_Gemella and (B) g_Streptococcus in samples from individual mice. The values are presented as the means ± SEM (n = 6–21). *P < 0.05, **P < 0.01 compared between control and B. breve M-16V diet OVA-allergic model mice. Spearman’s rank correlation between the relative abundance of (C) g_Gemella or (D) g_Streptococcus and mucin score shown in Fig 2B. Q-value is an FDR adjusted p-value.

Fig 7. Relationship between IL-5 expression in the lungs and fecal microbiota composition of each sample.

Fig 7

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Relative abundance of (A) g_Lactobacillus and (B) g_Dehalobacterium in samples from individual mice. The values are presented as the means ± SEM (n = 6–21). **P < 0.01 compared between control and B. breve M-16V diet OVA-allergic model mice. Spearman’s rank correlation between the relative abundance of (C) g_Lactobacillus or (D) g_Dehalobacterium and IL-5 mRNA expression shown in Fig 3B. Q-value is an FDR adjusted p-value.

Fig 8. Relationship between IL-13 expression in the lungs and fecal microbiota composition of each sample.

Fig 8

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Relative abundance of (A) g_Blautia and (B) g_Faecalibacterium in samples from individual mice. The values are presented as the means ± SEM (n = 6–21). *P < 0.05 compared between control and B. breve M-16V diet OVA-allergic model mice. Spearman’s rank correlation between the relative abundance of (C) g_Blautia or (D) g_Faecalibacterium and IL-13 mRNA expression shown in Fig 3C. Q-value is an FDR adjusted p-value.

Discussion

In the present study, we found that prenatal and postnatal supplementation with the B. breve M-16V combination was effective at reducing allergic inflammation in the airways of infant mice with OVA-induced allergic asthma delivered from a ROFA-exposed mother. Maternal supplementation with B. breve M-16V during pregnancy and breastfeeding changed the composition of gut microbiota; some genera correlated with several features of asthma including mucin score and Th2 cytokines including IL-5 and IL-13 in lungs.

Experimental and epidemiological studies have shown that air pollution factors, such as ROFA and DEP, are powerful adjuvants that promote allergic-type immune responses, which leads to skewed Th2 responses suggesting the transgenerational transmission of asthma risk after exposure to environmental particles during pregnancy [6]. In this study, the ROFA leachate contained high concentrations of metal elements, especially Ni, V, Zn, Co, Mn, Ca, and Cu [11]. Several of these metals were demonstrated to affect airway inflammation and physiologic responses in animal and epidemiologic studies [4, 35]. We previously demonstrated that the exposure of pregnant mice to an air pollutant surrogate (aerosolized ROFA leachate) exacerbated asthma-like airway Th2 inflammation in their offspring [11]. Furthermore, the maternal transfer of metals stimulated allergic responses and nickel may act as a hapten to induce allergic diseases [36]. Accordingly, the contribution of metals to ROFA-induced pulmonary injury during pregnancy was proven to be associated with increased immune cell infiltration into the lungs of offspring. However, in our previous study, aerosol exposure using a nickel sulfate solution at the same concentration and pH as the ROFA leachate did not reproduce this pro-asthmatic reaction [11], suggesting complex interactions between metals in the ROFA leachate might be required.

Probiotics are defined as live microorganisms, which, when administrated in adequate amounts, confer a health benefit to the host (FAO/WHO, 2002). Lactobacilli and bifidobacteria are the best-known probiotic candidates. Bifidobacteria are the major components of the human intestinal microbiota. Evidence is accumulating that specific probiotics including bifidobacteria counterbalance proallergic Th2-skewed immune responses [17, 37]. B. breve is a dominant bifidobacteria in the infant microbiota [38]. Strain M-16V, isolated from the fecal sample of a human baby [20], promoted the colonization of bifidobacteria and the formation of normal intestinal microbiota in low-birth-weight infants [22]. Furthermore, Hougee et al. demonstrated that, among six bacterial strains with potent antiallergic effects including four Bifidobacterium strains and two Lactobacillus strains, B. breve M-16V was the most effective at reducing allergic responses [39]. Here, we demonstrated that the oral administration of M-16V to mice during prenatal and postnatal periods improved asthma allergic inflammation in their offspring. Probiotics, especially M-16V, might facilitate the development of gut immune function and attenuate inflammation in preterm infants by changing gut microbial colonization [22]. Although recent studies have suggested that maternal supplementation with probiotics during pregnancy and breastfeeding may reduce the risk of eczema in their infants [40, 41], prenatal intervention alone did not affect the risk of eczema in infants [42]. According to this evidence, we chose maternal supplementation with probiotics during pregnancy and breastfeeding. Further studies are needed to evaluate the effect of M-16V supplementation in mothers on their offspring by nursing alone.

By analyzing the fecal microbiota composition of samples from neonatal mice, we found a lower proportion of Firmicutes in the OVA-allergic M-16V diet group compared with the OVA-allergic control diet group. A detailed analysis of the gut microbiota showed that Gemella was increased in the OVA-allergic M-16V diet group. Furthermore, this study showed it was inversely related to the mucin score. Sjödin et al. demonstrated that the inoculation of germ-free mice with Gemella decreased airway inflammation in their offspring [43]. Dehalobacterium was reported to be a key driver of sex-specific gut microbiota profiles [44], but its role in allergic responses is still unknown. The increased proportion of Streptococcus and Blautia may be negative regulators of the mucin score and IL-13, respectively. It is reported that Streptococcus and Blautia produce substances such as bacteriocins, enzymes, lactic acid, and fatty acids [45]. Furthermore, few bacterial genera, such as most members of Akkermansia, Allobaculum, Bacteroides, and Blautia produce short-chain fatty acids [46]. Previous studies reported that short-chain fatty acids reduced the risk of allergic airway disease and food allergy in offspring [47, 48]. Moreover, a high level of Faecalibacterium was strongly associated with a decreased level of IL-13. A decreased relative abundance of Faecalibacterium in early infancy was reported to be associated with increased asthma risk [44, 49]. These data suggest that in our study, higher levels of Blautia and Faecalibacterium had a strong relationship with decreased IL-13 expression in the lungs, which may be correlated with the reduced allergic responses by B. breve M-16V. Further investigation is required to determine the association between allergic responses and the microbiota and between the microbiota and each allergic-related parameter.

The pathogenesis of allergic asthma involves Th2-type cytokines, such as IL-4, IL-5, and IL-13 [13, 50]. IL-4 is a key cytokine that drives type-2 responses, IL-5 recruits eosinophils, and IL-13 is a pleiotropic cytokine produced predominantly by Th2 cells [51]. In our model, the gene expression levels of IL-5 and IL-13 in the lungs were significantly inhibited while IL-4 expression was intact in the OVA-allergic M-16V diet group compared with the control group. Moreover, the mucin score and expression of Muc5ac, which is localized to goblet cells in the surface epithelium and contributes to mucus production that correlates with asthma pathogenesis [52], were significantly improved in the M-16V diet group. Our previous report indicated that ROFA exposure in mothers did not enhance IL-4 production in OVA-allergic neonatal mice [11]. Additionally, recent accumulating studies demonstrated that type 2 innate lymphoid cells (ILC2) have a critical role in the pathogenesis of allergic asthma [53]. ILC2 mainly produced IL-5 and IL-13, but not IL-4, in an allergic asthma model [54]. Therefore, ILC2 might be suppressed by the administration of M-16V in our model. This evidence supports our data, which indicated that the gene expression of IL-4 was not significantly changed by the M-16V diet. We also measured protein levels (IL-4, IL-5, and IL-13) in BALF, however, there was no significant difference in these cytokines. We expect deviation of time course between gene expressions in lungs and cytokine productions in BALF. Further investigation including the role of ILC2 and the time point of analysis in the M-16V diet in allergic inflammation is necessary.

In summary, we demonstrated that the administration of B. breve M-16V to mothers during pregnancy and breastfeeding prevented the development of OVA-induced asthma-like allergic inflammation accelerated by air pollution ROFA in their offspring by inhibiting Th2 immune responses and changes in their gut microbiota. Of note, B. breve M-16V is safe for use as a probiotic in humans including premature infants and pregnant women [23]. Thus, additional studies of the preventive potential of M-16V for allergic asthma are warranted, and M-16V might ultimately have clinical applicability.

Supporting information

S1 Fig. Lung histopathologic analysis of OVA allergic neonates from mothers exposed to PBS (no ROFA) on a control (AIN-93G) or M-16V diet.

(A) Lung sections were stained with PAS to visualize mucus production at day 30 of life. (B) Quantitative analysis of mucus production using a scoring system of 1–4 detailed in the Materials and methods. The values are presented as the means ± SEM (n = 6–8).

(TIF)

Click here for additional data file. (3.9MB, tif)
S2 Fig. Lung histopathologic analysis of OVA allergic neonates from PBS- or ROFA-exposed mothers on a control (AIN-93G) diet.

(A) Lung sections were stained with PAS to visualize mucus production at day 30 of life. (B) Quantitative analysis of mucus production using a scoring system of 1–4 detailed in the Materials and methods. The values are presented as the means ± SEM (n = 6–7). *P < 0.05 compared with OVA-sensitized and -exposed neonates from mothers without ROFA exposure.

(TIF)

Click here for additional data file. (3.8MB, tif)
S3 Fig. Cytokine production from the BALF supernatant.

BALF was collected 48 h after the final aerosol challenge, and BALF supernatant was collected after the centrifugation of samples at 800 ×g for 5 min. Cytokine productions of IL-4, IL-5, and IL-13 from BALF supernatants were measured by ELISA. The values are presented as the mean ± SEM (n = 6–21).

(TIF)

Click here for additional data file. (267KB, tif)

Acknowledgments

We thank Ms. Reiko Masuda, Ms. Hisayo Nishikawa, and Mr. Ryo Misawa (Nara Medical University) for their assistance. We thank J. Ludovic Croxford, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.

Data Availability

DNA sequences corresponding to the 16S rRNA gene data have been deposited in DDBJ under accession number DRA010221.

Funding Statement

This study was supported by research grants from Morinaga Milk Industry Co., Ltd. The funder provided support in the form of salaries for A. Hiraku, K. Kato and N. Iwabuchi, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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Decision Letter 0

Heinz Fehrenbach

20 Apr 2020

PONE-D-20-05759

Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol

PLOS ONE

Dear Dr. Ito,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Two experts from the field and I myself have reviewed your manuscript. Although the study is in general interesting and well performed, there are several points that need to be clarified in a revised version of the manuscript. In particular, but not excluively, the lack of control groups not being exposed to ROFA would have been desirable. This has also implications for the title of your manuscript. Otherwise, the manuscript cannot make any convincing conclusion about the role of ROFA in this setting. Further, the statistics used need to be partly revised and several details should be added in a revised version of the manuscript. A detailed point-by-point response to all the comments will be mandatory.

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Additional Editor Comments (if provided):

In additional to the reviewers' comments, I raise the following points:

- the effects in the OVA models after ROFA exposure during pregancy are very mild; so if ROFA aggravates the response of neonatal mice to OVA, I wonder whtethger there is any significant effect in mice that were not exposed to ROFA

- only few cytokines were analysed / presented and only at the transcriptional level; it would be important to also give the protein levels in BALF

- IL-4 is of majopr importance for eosinophilc inflammation; however, expression of IL-4 was not significantly changed by the diet; please comment

- although the methodology of mucin scoring was referenced, it would be helpful to briefly state how the analysis was performed and statistically analysed

- histopathological overview micrographs should be complemented by high power micrographs to be able to assess the inflammaion

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I have following recommendations and comments on the manuscript with the title "Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol":

1. The relevance of exposing the pregnant mice with ROFA in the mouse model is hardly comprehensible. Data from mice which were not ROFA exposed is not included. It seems that the Th2 immune reaction in the offspring is induced by OVA exposure rather than additional ROFA treatment of the mothers. The authors should explicitely figure out why ROFA exposure of the pregnant animals is of importance for their manuscript. If possible, they should include a short comparative data set of Th2 inflammation markers of OVA-exposed offsprings from ROFA and not ROFA +/- M-16V treated mothers.

2. In the description of the mouse model it´s unclear if M-16V diet of the mothers was continued throughout the nursing time. If so, the authors should at least shortly discuss a possible effect of M-16V on the offsprings just by nursing.

3. In figure 6 and 7 correlation calculations were performed on the incidence of bacterial species with Th2 cytokine mRNA expression. However, the information is missing if or not IL-5 expression level is also correlated e.g. with Blautia species and IL-13 e.g. with Lactobaccillus spec.

4. In figure 2 and 5 please correct the wording "mucin score" at the Y- and X-axis, respectively.

Reviewer #2: In the present manuscript titled „ Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol” Terada-Ikeda et al examined the effect of the bifidobacterial strain M-16V fed to pregnant mice on the severity of allergic airway disease in offspring mice.

The study is well structured, concise and contains interesting findings. However, I have several major points regarding the experimental-setup.

X the title is misleading as it gives emphasis on pollution expose of mothers, which was not investigated in this study - all animals were treated with ROFA. I understand that a ROFA-treated group stands on its own, however, a control group (no ROFA exposure) would have largely benefited the study, and authors should discuss why no such group was included. In line with this, the first page of the discussion section can be omitted as it deals in detail with the effects of ROFA treatment.

X the samples sizes applied are not clear. They are not given in the materials and methods section. In the Figure legends they vary between parameters, namely, n=4-8 in Fig 1-3 and n=6-21 in Figures displaying results from microbiota analyses. Why is there such a difference in sample sizes? It seems not all parameters were measured for all animals?

X How were mice housed? In single cages or in groups? For microbiota studies cage-effects are widely known.

X stats applied are not always appropriate. It reads that ANOVA was used throughout, however, often microbiota data is not normally distributed (see e.g. Streptococcus). Furthermore, correction for multiple testing has to be applied.

X what was the diet of neonates? Initially, I thought they were breast-fed, however, reading the first sentence in the discussion section I was confused as authors speak about postnatal supplementation of the probiotic. Nothing is given in the material and methods section.

X more information on library preparations should be given. Previous work is referenced, yet major information should be included here as well, such as the kit used for DNA extraction, what primers were used, … .

X no link for 16S rRNA-gene data download is given.

Minor points

X what was the sex of offsprings?

X L263: p-value for Actinos = 0.14, so it is not significant.

X L265: according to Fig 5 Gemella comprises only ~1% of total bacteria, so it is not predominant.

X L266: mucus microbiota was not investigated, but fecal samples were used for analyses.

X 273: Blautia is not a major butyrate producer in gut microbiota – only some specific strains might produce this compound.

X 280: omit “led-to” – it implies causality, which was not investigated here. In general, care should be taken with the wording throughout the study as only associations were obtained.

X some parts of the discussion read like a commercial for M-16V.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 11;15(9):e0238923. doi: 10.1371/journal.pone.0238923.r002

Author response to Decision Letter 0

17 Jul 2020

In additional to the reviewers' comments, I raise the following points:

We do appreciate the comments and suggestions by the editor, and we have addressed each with additional comments and experiments.

- the effects in the OVA models after ROFA exposure during pregnancy are very mild; so if ROFA aggravates the response of neonatal mice to OVA, I wonder whether there is any significant effect in mice that were not exposed to ROFA.

Here, we first assessed whether ROFA aggravates the response of neonatal mice to OVA. We confirmed that ROFA exposure in mothers aggravates the response of neonatal mice by mucin score and gene expressions of IL-5 and IL-13 (S2 Fig). We have also previously reported that offspring from mothers exposed to ROFA during pregnancy induced robust pathologic changes of airway inflammation including mucin scoring (Ref 11).

Moreover, we compared the response of neonatal mice to OVA without ROFA exposure between control (AIN-93G) and M-16V diet. As shown in S1 Fig, there was no significant effect in mice that were not exposed to ROFA between control (AIN-93G) and M-16V diet.

- only few cytokines were analyzed / presented and only at the transcriptional level; it would be important to also give the protein levels in BALF

We measured protein levels (IL-4, IL-5, and IL-13) in BALF. However, there was no significant difference in these cytokines (S3 Fig). We expect deviation of time course between gene expressions in lungs and cytokine productions in BALF. We mentioned it in “Discussion” (L364-367).

- IL-4 is of major importance for eosinophilc inflammation; however, expression of IL-4 was not significantly changed by the diet; please comment.

We totally agree that IL-4 is one of major Th2 cytokines including eosinophilic inflammation. Our previous report indicated that ROFA exposure in mothers did not enhance IL-4 production in OVA-allergic neonatal mice (Ref 11). Here, we also demonstrate that ROFA exposure in mothers aggravated the gene expressions of IL-5 and IL-13, not IL-4, in lungs of neonatal mice (S2 Fig), and that those of IL-5 and IL-13, not IL-4, were significantly lower in the OVA-allergic M-16V diet group compared with the control diet (AIN-93G) group. Also, there was no significant effect in mice that were not exposed to ROFA between control (AIN-93G) and M-16V diet (S1 Fig).

Additionally, recent accumulating studies have demonstrated that type 2 innate lymphoid cells (ILC2) have a critical role in the pathogenesis of allergic asthma (Ref. 53). ILC2 mainly produced IL-5 and IL-13, but not IL-4 in an allergic asthma model (Ref. 54). This evidence supports our data, which indicated that the gene expression of IL-4 was not significantly changed by the M-16V diet. We added the above comment to “Discussion” (L357-368).

- although the methodology of mucin scoring was referenced, it would be helpful to briefly state how the analysis was performed and statistically analyzed.

The methodology of mucin scoring was mentioned in “Materials and methods” (L125-128).

- histopathological overview micrographs should be complemented by high power micrographs to be able to assess the inflammation.

High power histological micrographs were added to Figure 2A.

Reviewer #1:

I have following recommendations and comments on the manuscript with the title "Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol":

We do appreciate the comments and suggestions by Reviewer #1, and we have addressed each with additional comments and experiments.

1. The relevance of exposing the pregnant mice with ROFA in the mouse model is hardly comprehensible. Data from mice which were not ROFA exposed is not included. It seems that the Th2 immune reaction in the offspring is induced by OVA exposure rather than additional ROFA treatment of the mothers. The authors should explicitely figure out why ROFA exposure of the pregnant animals is of importance for their manuscript. If possible, they should include a short comparative data set of Th2 inflammation markers of OVA-exposed offsprings from ROFA and not ROFA +/- M-16V treated mothers.

We totally agree that our previous manuscript was not enough to explain why ROFA exposure of the pregnant animals is of importance. Here, we first assessed whether ROFA aggravates the response of neonatal mice to OVA. We confirmed that ROFA exposure in mothers aggravates the response of neonatal mice by mucin score and gene expressions of IL-5 and IL-13 (S1 Fig). We have also previously reported that offspring from mothers exposed to ROFA during pregnancy induced robust pathologic changes of airway inflammation including mucin scoring (Ref 11).

Moreover, we compared the response of neonatal mice to OVA without ROFA exposure between control (AIN-93G) and M-16V diet. As shown in S2 Fig, there was no significant effect in mice that were not exposed to ROFA between control (AIN-93G) and M-16V diet.

2. In the description of the mouse model it´s unclear if M-16V diet of the mothers was continued throughout the nursing time. If so, the authors should at least shortly discuss a possible effect of M-16V on the offsprings just by nursing.

M-16V diet of the mothers was continued throughout the nursing time until analysis of offsprings. So, we now discuss a possible effect of M-16V on the offsprings just by nursing (L325-327).

3. In figure 6 and 7 correlation calculations were performed on the incidence of bacterial species with Th2 cytokine mRNA expression. However, the information is missing if or not IL-5 expression level is also correlated e.g. with Blautia species and IL-13 e.g. with Lactobaccillus spec.

We just pick up the results which were significantly correlated with between gene expression in lungs and bacteria genera in fecus. IL-5 expression level was not correlated with Blautia species, and IL-13 expression level was not correlated with Lactobaccillus spec. It is now mentioned in “Result” (L279-281).

4. In figure 2 and 5 please correct the wording "mucin score" at the Y- and X-axis, respectively.

Thank you for pointing it out. We corrected the wording “mucin score”.

Reviewer #2:

In the present manuscript titled „ Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol” Terada-Ikeda et al examined the effect of the bifidobacterial strain M-16V fed to pregnant mice on the severity of allergic airway disease in offspring mice.

The study is well structured, concise and contains interesting findings. However, I have several major points regarding the experimental-setup.

We do appreciate the comments and suggestions by Reviewer #2, and we have addressed each with additional comments and experiments.

1. The title is misleading as it gives emphasis on pollution expose of mothers, which was not investigated in this study - all animals were treated with ROFA. I understand that a ROFA-treated group stands on its own, however, a control group (no ROFA exposure) would have largely benefited the study, and authors should discuss why no such group was included. In line with this, the first page of the discussion section can be omitted as it deals in detail with the effects of ROFA treatment.

We totally agree that the title is misleading in our previous manuscript because all animals were treated with ROFA. The editor and all reviewers pointed out that there was no control group (no ROFA exposure). Here, we first assessed whether ROFA aggravates the response of neonatal mice to OVA. We confirmed that ROFA exposure in mothers aggravates the response of neonatal mice by mucin score and gene expressions of IL-5 and IL-13 (S2 Fig). We have also previously reported that offspring from mothers exposed to ROFA during pregnancy induced robust pathologic changes of airway inflammation including mucin scoring (Ref 11).

Moreover, we compared the response of neonatal mice to OVA without ROFA exposure between control (AIN-93G) and M-16V diet. As shown in S1 Fig, there was no significant effect in mice that were not exposed to ROFA between control (AIN-93G) and M-16V diet.

2. The samples sizes applied are not clear. They are not given in the materials and methods section. In the Figure legends they vary between parameters, namely, n=4-8 in Fig 1-3 and n=6-21 in Figures displaying results from microbiota analyses. Why is there such a difference in sample sizes? It seems not all parameters were measured for all animals?

We analyzed again the data (BAL, Real-time PCR, Mucin score) using n=6-21 in Figures displaying results from microbiota analyses. As shown in figure 2-4, we indicate new data (n=6-21), and the result trend does not change compared with previous data (n=4-8).

3. How were mice housed? In single cages or in groups? For microbiota studies cage-effects are widely known.

Each mother was house with her offspring in single cage. It was mentioned in “Materials and methods” (L90-92).

4. Stats applied are not always appropriate. It reads that ANOVA was used throughout, however, often microbiota data is not normally distributed (see e.g. Streptococcus). Furthermore, correction for multiple testing has to be applied.

The analyses of gut microbiota without the calculation of a false discovery rate (FDR) were performed using SPSS version 23.0 statistical software (IBM Corp., Armonk, NY). Intergroup differences were analyzed using the Mann-Whitney U-test and correlation analysis between gut bacteria and Th2 responses in the lungs were performed by Spearman’s correlation coefficient. Results were adjusted by FDR using the Benjamini and Hochberg method in R software version 3.6.0. (L196-202).

5. What was the diet of neonates? Initially, I thought they were breast-fed, however, reading the first sentence in the discussion section I was confused as authors speak about postnatal supplementation of the probiotic. Nothing is given in the material and methods section.

We are sorry for confusing. They were breast-fed. We now mention it in “Materials and methods” (L91).

6. More information on library preparations should be given. Previous work is referenced, yet major information should be included here as well, such as the kit used for DNA extraction, what primers were used.

We added more information related to library preparation including DNA extraction method and primer sequencings to "Materials and methods" (L143-176).

7. No link for 16S rRNA-gene data download is given.

DNA sequences corresponding to the 16S rRNA gene data have been deposited in DDBJ under accession number DRA010221 (L186-188).

8. What was the sex of offsprings?

All offsprings (both male and female) was used. It is also mentioned in “Materials and methods” (L91).

9. L263: p-value for Actions = 0.14, so it is not significant.

We deleted the sentence about Actinobacteria.

10. L265: according to Fig 5 Gemella comprises only ~1% of total bacteria, so it is not predominant.

We deleted the sentence together with comment #11.

11. L266: mucus microbiota was not investigated, but fecal samples were used for analyses.

We deleted the sentence together with comment #10.

12. L273: Blautia is not a major butyrate producer in gut microbiota – only some specific strains might produce this compound.

We agree that Blautia is not a major butyrate producer in gut microbiota, so we changed the sentence about Blautia according to Ref. 45 & 46(L337-340).

13. L280: omit “led-to” – it implies causality, which was not investigated here. In general, care should be taken with the wording throughout the study as only associations were obtained.

We omitted “led to”, and changed the sentence (L346).

14. Some parts of the discussion read like a commercial for M-16V.

We deleted some sentences which read like a commercial for M-16V in Discussion.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (27.7KB, docx)

Decision Letter 1

Heinz Fehrenbach

11 Aug 2020

PONE-D-20-05759R1

Bifidobacterium breve M-16V prevents allergic airway inflammation accelerated by prenatal exposure to an air pollutant aerosol

PLOS ONE

Dear Dr. Ito,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Some final comments:

1. Lines 337-339: The sentence sounds incomplete. Please check.

2. Line 347: The wording "improved allergic responses" can be misleading. Better use "reduced allergic responses".

Reviewer #2: most of my comments were adressed. However, I feel it reads a bit complicated now, so I have the following suggestions:

1. Change the title to a more simplistic one - it is hard to get the message

2. The additional first paragraphs in the results section confuse the reader on what was done in the following experiments. State clearly which mice where used for experiments and what exposure was applied. Say that only ROFA exposed mice were used later and that this was the focus.

3. Accordingly, Figure 1 is misleading as it suggests all groups have been investigated throughout the experiment - please communicate the experimental setup clearly to the reader.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 11;15(9):e0238923. doi: 10.1371/journal.pone.0238923.r004

Author response to Decision Letter 1

20 Aug 2020

Reviewer #1:

Some final comments:

We again appreciate the comments and suggestions by Reviewer #1, and we have addressed each with additional comments.

1. Lines 337-339: The sentence sounds incomplete. Please check.

Thank you for the suggestion. The sentence was incomplete. We changed the sentence as follows; “It is reported that Streptococcus and Blautia produce substances such as bacteriocins, enzymes, lactic acid, and fatty acids.”

2. Line 347: The wording "improved allergic responses" can be misleading. Better use "reduced allergic responses".

We now use "reduced allergic responses" as suggested.

Reviewer #2:

Most of my comments were addressed. However, I feel it reads a bit complicated now, so I have the following suggestions:

We again appreciate the comments and suggestions by Reviewer #2, and we have addressed each with additional comments.

1. Change the title to a more simplistic one - it is hard to get the message

Thank you for the suggestion. We changed the title as follows to send the message for readers; “Maternal supplementation with Bifidobacterium breve M-16V prevents their offspring from allergic airway inflammation accelerated by the prenatal exposure to an air pollutant aerosol”.

2. The additional first paragraphs in the results section confuse the reader on what was done in the following experiments. State clearly which mice where used for experiments and what exposure was applied. Say that only ROFA exposed mice were used later and that this was the focus.

We modified Figure 1 to state clearly which mice where used for experiments and what exposure was applied. Also, we now say that only ROFA exposed mice were used later and that this was the focus in this study (L221).

3. Accordingly, Figure 1 is misleading as it suggests all groups have been investigated throughout the experiment - please communicate the experimental setup clearly to the reader.

We modified Figure 1 together with comment #2 to communicate the experimental setup clearly to the reader.

Attachment

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Decision Letter 2

Heinz Fehrenbach

27 Aug 2020

Maternal supplementation with Bifidobacterium breve M-16V prevents their offspring from allergic airway inflammation accelerated by the prenatal exposure to an air pollutant aerosol

PONE-D-20-05759R2

Dear Dr. Ito,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Heinz Fehrenbach

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

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Acceptance letter

Heinz Fehrenbach

2 Sep 2020

PONE-D-20-05759R2

Maternal supplementation with Bifidobacterium breve M-16V prevents their offspring from allergic airway inflammation accelerated by the prenatal exposure to an air pollutant aerosol

Dear Dr. Ito:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Lung histopathologic analysis of OVA allergic neonates from mothers exposed to PBS (no ROFA) on a control (AIN-93G) or M-16V diet.

    (A) Lung sections were stained with PAS to visualize mucus production at day 30 of life. (B) Quantitative analysis of mucus production using a scoring system of 1–4 detailed in the Materials and methods. The values are presented as the means ± SEM (n = 6–8).

    (TIF)

    Click here for additional data file. (3.9MB, tif)
    S2 Fig. Lung histopathologic analysis of OVA allergic neonates from PBS- or ROFA-exposed mothers on a control (AIN-93G) diet.

    (A) Lung sections were stained with PAS to visualize mucus production at day 30 of life. (B) Quantitative analysis of mucus production using a scoring system of 1–4 detailed in the Materials and methods. The values are presented as the means ± SEM (n = 6–7). *P < 0.05 compared with OVA-sensitized and -exposed neonates from mothers without ROFA exposure.

    (TIF)

    Click here for additional data file. (3.8MB, tif)
    S3 Fig. Cytokine production from the BALF supernatant.

    BALF was collected 48 h after the final aerosol challenge, and BALF supernatant was collected after the centrifugation of samples at 800 ×g for 5 min. Cytokine productions of IL-4, IL-5, and IL-13 from BALF supernatants were measured by ELISA. The values are presented as the mean ± SEM (n = 6–21).

    (TIF)

    Click here for additional data file. (267KB, tif)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (27.7KB, docx)
    Attachment

    Submitted filename: Response to reviewer-2.docx

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    Data Availability Statement

    DNA sequences corresponding to the 16S rRNA gene data have been deposited in DDBJ under accession number DRA010221.

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