Introduction

Atopic dermatitis (AD) is an allergic skin disease [1]. High serum immunoglobulin (Ig) E levels are a notable feature in some AD patients [2]. The interaction of genetic and environmental factors influences the normal immune responses and thus is associated with AD [3]. Although the pathogenesis of AD remains to be elucidated, accumulating studies have evidenced that T helper (Th) 2-type immune responses are prevalent in the acute stage of AD while Th1- and Th17-type immune responses are predominant the development of AD at the chronic stage [4, 5]. Additionally, immune responses, inflammatory cells recruitment, and inflammatory infiltration play key roles in AD pathogenesis [6].

The gut is the important immune organ for animals and humans, and the gut microbiota includes up to 1014 bacterial cells [7, 8]. Immune homeostasis is composed of the interaction with epithelial cells, antigen processing cells, lymphocytes in the gut, and intestinal microorganisms provoke the differentiation of regulatory T cells (Tregs) to increase immune tolerance through this interaction [9]. Therefore, the balance in the micro-ecology system of gut microbiota is important for immune responses [10]. Recent studies have investigated that an imbalance of gut microbiota relates to the development of allergic diseases including AD [11,12,13]. However, causes of the differences are difficult to determine because the gut microbiota is affected by many complex factors.

To alleviate AD symptoms, many approaches including probiotics treatment, have been used in both animal and clinical studies. Probiotics regulate intestinal flora and have good benefits for gut immunity, thereby alleviating AD symptoms. For instance, Lactobacillus sakei WIKIM30, a Gram-positive bacterium, ameliorates AD symptoms by inducing Tregs differentiation and altering the gut microbiota in a mouse model [14]. Additionally, supplement with a probiotic mixture including B. longum CECT 7347, B. lactis CECT 8145, and L. casei CECT 9104 significantly decreases Scoring Atopic Dermatitis (SCORAD) index and the use of topical steroids in patients with AD. However, the mechanism is not fully understood [15].

In this study, 2,4-dinitrofluorobenzene (DNFB) was used to induce AD symptoms in mice. This model was used to evaluate the effect of six strains of B. adolescentis on AD symptoms and immune responses. A study has demonstrated that in chronic asthma patients, although the proportion of Bifidobacterium decreased, the B. adolescentis species prevailed [16]. While previously study has demonstrated B. adolescentis CCFM 669 and CCFM667 alleviated loperamide-induced constipation through the increase in propionic and butyric acids in BALB/c mice [17]. Additionally, some studies have reported that commensal intestinal bacteria were able to stimulate the generation of Foxp3+ Tregs, frequently in a SCFAs-dependent mechanism [18]. Based on these debates, we proposed a hypothesis that B. adolescentis might attenuate disease symptoms through regulating gut microbiota and affecting gut microbiota-derived SCFAs production. In addition, the correlation between clinical manifestation, immune responses, and gut microbial alteration was evaluated to explore possible alleviation mechanisms.

Materials and methods

Bifidobacterium adolescentis strains

Six B. adolescentis strains FJSYC5M10 (Ad1), FJSYC6M8 (Ad2), FJSYC7M3 (Ad3), FAHBZ6M2 (Ad4), FHNXY72M2 (Ad5), and FAHBZ2M5 (Ad6) derived from Culture Collection of Food Microorganisms of Jiangnan University (Wuxi, China). Bifidobacteria adolescentis were isolated from the healthy volunteer feces or animal feces. All strains were consecutively reactivated in an anaerobic incubator (Electrotek AW500SG, England) at least three times using 2% (v/v) inoculum in MRS broth with 0.5% l-cysteine (w/v) at 37 °C (Supplementary material).

Animals and treatment

Six-week-old, female, and specific-pathogen-free grade C57bl/6 mice were kept in a facility under light cycle (12 h light/12 h dark), temperature (25 °C), and humidity level (50%). The study protocol was approved by the Ethics Committee of Jiangnan University, China (JN. No20180115c2600531 [5]).

All mice were fed standard chow and water ad libitum. Mice were divided into the following groups: control group (fed with the vehicle), DNFB group (fed with the vehicle), Keto group (fed with ketotifen, 1 mg/kg·b.w. (bodyweight)) and B. adolescentis group (fed with B. adolescentis) and the detailed randomization process was in Supplementary material. AD symptoms were induced by DNFB as Sasso et al. described and modified [19]. 0.5% DNFB solution (substrate solution: acetone/olive oil (4:1, v/v)) was painted on the shaved dorsal skin and left ear of mice on day 1. After painting, mice were painted on days 5, 8, 11, 14 with 0.2% DNFB solution. Mice were only treated with a substrate solution in the control group. The Bifidobacterium spp. suspension of 0.2 ml (1 × 109 CFU) was orally administrated once a day for 3 weeks, and Fig. 1a showed the experimental flow chart.

Fig. 1
figure 1

Effect of Bifidobacterium adolescentis on ear thickness. a Animal experimental schedule. b Effects of B. adolescentis on ear thickness in AD mice (n = 5/group)

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Ear thickness determination

A digital vernier caliper was used to determine the thickness at the middle sections of the ear.

Measurement of skin thickness

The dorsal skin was stained with haematoxylin and eosin. Photomicrographs and skin thickness were taken at a fixed magnification of 400× by the digital scanner.

Mast cells stain

Mast cells were stained by toluidine blue, and then the number of mast cells was calculated.

Measurement of immune indicators

After sacrifice, skin tissues and serum samples were collected and determined by enzyme-linked immunosorbent assay kits (Senbeijia, Nanjing, China).

Flow cytometry analysis

After sacrifice, referring to the protocol, single-cell suspension of spleen was prepared for CD4, CD25 and Foxp3 intracellular staining by mouse regulatory T cell staining kit #1 (Invitrogen Corporation, Carlsbad, CA, USA).

16S rRNA amplicon sequencing and SCFAs metabolism

To access changes in gut flora, the total DNA of feces was collected by FastDNA Spin Kits (MP Biomedicals, Santa Ana, USA). The V3–V4 region was amplified and sequenced at the Illumina MiSeq platform (Illumina, Santiago, CA, USA) as described previously [20] (Supplementary material). The microbial biomarkers were identified by linear discriminant analysis (LDA) effect size (LEfSe) method. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was performed to analyze the functional genes profiles of gut microbiota based on 16S information [21]. The functional genes predictive analysis was performed by the Galaxy website (Supplementary material).

SCFAs were measured by GCMS, referring to Mao et al. [22]. SCFAs concentrations were determined according to the external standard method (Supplementary material).

Statistical analysis

The statistical analysis was performed with SPSS 24.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, USA). Data represent the mean ± SD (n = 5/group). The Kruskal–Wallis tests (non-parametric analysis) was used to compare the statistical difference among all groups, followed by Dunn’s multiple comparisons tests (*P < 0.05, **P < 0.01, ***P < 0.001 vs DNFB group). To examine the relationship between parameters associated with AD and gut microbial alteration, a principal component analysis (PCA) was carried out by XLSTAT (Addinsoft, Paris, France). The network correlation between variations was performed by R (version 3.5.1) and Gephi (version 0.9.2) [23].

Results

Bifidobacteria adolescentis alleviated AD-like clinical symptom

The inhibition effect of B. adolescentis on clinical symptoms in mice with AD was shown in Fig. 1b. Bifidobacteria adolescentis Ad1, Ad3, and Ad6 decreased the ear thickness versus the DNFB group, but B. adolescentis Ad2, Ad4, and Ad5 could not alleviate ear swelling. We next performed the histological analysis. Skin thickness was improved by oral administration of B. adolescentis Ad1 and Ad3 (Fig. 2a, b), besides, the recruitment of mast cells was inhibited by Ad1 treatment compared to the DNFB group (Fig. 2c, d). In summary, these results demonstrated that B. adolescentis Ad1 and Ad3 treatments effectively alleviated AD-like clinical symptoms.

Fig. 2
figure 2

Effects of Bifidobacterium adolescentis treatments on symptoms of atopic dermatitis. a Dorsal skin sections were stained, Scale bar = 200 μm, original magnification = ×400. b Changes in skin thickness (n = 5/group). c Mast cells were stained (red arrowhead), Scale bar = 200 μm (×5), 50 μm (×20), original magnification = ×400. d Mast cell number was evaluated (n = 5/group)

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Bifidobacteria adolescentis promoted Tregs differentiation in the spleen

Tregs differentiation plays an important role in the acquisition of immunosuppression. To investigate whether B. adolescentis administration affected immunomodulation in the spleen, regulatory T cells (Tregs) population were measured by flow cytometry (Fig. 3a). Ad1, Ad3, and Ad5 treatments significantly increased Tregs ratio in spleen versus the DNFB group (Fig. 3b). The other three strains also promoted the Tregs differentiation although there was no statistical difference. The results suggested that oral administration of B. adolescentis Ad1, Ad3, and Ad5 induced Tregs differentiation, thereby resulting in suppressing the Th2-dominant immune response in AD.

Fig. 3
figure 3

Tregs ratio in the spleen. a The percentages of Tregs were analyzed by flow cytometry. b Tregs ratio was measured (n = 5/group)

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Bifidobacteria adolescentis suppressed serum IgE and regulated cytokines production

The levels of serum IgE and cytokines related to AD were determined. Bifidobacteria adolescentis Ad1, Ad4, Ad5, and Ad6 significantly reduced serum IgE levels compared to the DNFB group (Fig. 4a). All strains decreased IL-4 levels in dorsal skin except for Ad3, although there was no statistical significance in these groups (Fig. 4b). Compared to the DNFB group, B. adolescentis Ad5 and Ad6 treatments reduced the expression levels of IL-13, but there was no statistical significance (Fig. 4c). Bifidobacteria adolescentis Ad1 and Ad4 treatments decreased IL-5 levels versus the DNFB group (Fig. 4d). In addition, oral administration of Ad1 and Ad2 decreased the expression levels of macrophage-derived chemokine (MDC/CCL22) versus the DNFB group (Fig. 4e). Bifidobacteria adolescentis Ad1–Ad3 significantly increased IFN-γ and IL-10 levels (Fig. 4f, g). These results showed that B. adolescentis had variable effects on immune responses in DNFB-induced AD mice.

Fig. 4
figure 4

Effects of B. adolescentis on cytokine levels and serum IgE (n = 5/group)

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Bifidobacteria adolescentis affected the gut microbiota diversity in AD mice

To determine whether B. adolescentis can influence the gut microbiota composition, the fecal flora obtained from mice were analyzed by high-throughput sequencing. The mice from the DNFB group showed dramatic shifts in gut microbial composition and structure versus the control group (Fig. 5a). Firmicutes was reduced in the DNFB group, and the ratio of Firmicutes/Bacteroidetes (F/B), a signature of gut dysbiosis, was decreased in the DNFB group (Fig. 5b). Treatment with B. adolescentis strains prevented the DNFB-induced increases in Bacteroidetes while increased the proportion of Firmicutes, and B. adolescentis Ad2, Ad4, Ad5, and Ad6 treatments significantly increased the F/B ratio versus the DFNB group (Fig. 5b).

Fig. 5
figure 5

Analysis of gut microbiota composition. a The gut microbial composition at the phylum level. b Changes in the F/B ratio (n = 4–5/group). c α diversity indicated by Chao1 and Shannon indices (n = 4–5/group). d β diversity based on Bray–Curtis similarity

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Additionally, the Shannon and Simpson diversity index were evaluated to exhibit gut microbial diversity (Fig. 5c). Treatment with B. adolescentis strains increased the Chao1 index, particularly, B. adolescentis Ad1 significantly increased the Chao1 index versus the DNFB group. However, the Shannon index was no differences between all groups. The beta diversity showed the differences in gut microbial composition among all groups (Fig. 5d).

Bifidobacteria adolescentis regulated the gut microbiota composition and SCFAs metabolism

To further explore changes in the gut microbiota, LEfSe analysis was performed. The taxonomic cladogram and tag exhibited different taxa in the DNFB group compared to all other groups (Fig. 6a, b, Fig. S2). Pseudomonas, Sutterella, Enterococcus, Dorea, and Pediococcus were the core microbiome in the DNFB group compared to all other groups. Some selected different genera were shown in Fig. 6c, the relative abundance of Enterococcus was significantly increased in DNFB group, while this was restored by B. adolescentis Ad1, A3, A4, and Ad5 treatments. Bifidobacteria adolescentis Ad1 treatment decreased the proportion of Dorea. Additionally, the reduction in Lactobacillus was restored by B. adolescentis Ad1 treatment.

Fig. 6
figure 6

Differential gut microbiota among all groups. a LEfSe comparison of microbiota in all groups. b The differential genus in the DNFB group compared to Ad1–Ad6 groups. c The relative abundance of Enterococcus, Dorea, and Lactobacillus

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To reveal changes in the metabolism of gut microbiota, the colonic SCFAs were determined. Acetic acid, propionic acid, and butyric acid were decreased while isovaleric acid was increased in DNFB group (Fig. 7a). Treatment with B. adolescentis Ad1 increased propionic and butyric acids but decreased isovaleric acid, and could not increase acetic acid versus the DNFB group. Bifidobacteria adolescentis Ad4 and Ad5 treatments also decreased isovaleric acid versus the DNFB group.

Fig. 7
figure 7

Changes in SCFAs and KEGG pathways in the control, DNFB, and Ad1 groups. a Changes in SCFAs (n = 5/group). b PCA score scatter plot of clinical, immune, and intestinal measures of the six strains. c Differential KEGG pathways between the control and DNFB groups (Welch’s t test, two-sided, P < 0.05); Differential KEGG pathways between the DNFB and Ad1 groups (Welch’s t test, two-sided, P < 0.05, effect size < 1.10). d Changes in differential KEGG pathways

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Bifidobacteria adolescentis modulated functional modules of the gut microbiota

To explore the effects of B. adolescentis on the physiological functions of gut microbiota, PICRUSt analysis was performed to analyze and predict the composition of the functional genes in metabolic pathways. First, based on clinical and immune indicators, and changes in gut microbiota, a PCA analysis was performed to select the possible effective strain for further research (Fig. 7b). The distance of points represented the similarity among groups. Bifidobacteria adolescentis Ad1, Ad2, and Ad3 located closer to the control group than other B. adolescentis strains. Therefore, we selected B. adolescentis Ad1 to conduct PICRUSt analysis. At the P < 0.05 from the t test (two-sided), 11 different functional categories were identified between the control and DNFB groups (Fig. 7c). Pathways involved in C5-branched dibasic acid metabolism, phenylpropanoid biosynthesis, and valine, leucine and isoleucine biosynthesis were upregulated in DNFB group, but other eight pathways, such as polycyclic aromatic hydrocarbon degradation, fructose and mannose metabolism, and phosphotransferase system were upregulated in the control group. The differential pathways between the DNFB and Ad1 groups were exhibited in Fig. 7c. Bifidobacteria adolescentis Ad1 treatment also upregulated fructose and mannose metabolism versus the DNFB group, and pathway involved in fatty acid biosynthesis of gut microbiota was upregulated. Additionally, antigen processing and presentation and nucleotide-binding oligomerization domain-like receptor signaling pathways were significantly upregulated by B. adolescentis Ad1 treatment, although it was no statistical significance in the control and DNFB groups (Fig. S3).

Correlation between clinical symptoms, immune responses, and gut microbial alteration

To reveal the relationship among variations related to pathological and immune indicators, SCFAs production, and gut microbiota, the interaction network analysis was performed. The differential taxa were selected based on LEfSe analysis in the Control, DNFB, Ad1, Ad2, and Ad3 groups (Fig. 6a and Fig. S2). Figure 8a showed that skin thickness and mast cells were positively related to IL-5, CCL22, Rikenella, and Dorea but negatively related to Tregs, IFN-γ, propionic acid, butyric acid, and Lactobacillus (P < 0.05, r > 0.3 or r < − 0.3). Obviously, clinical symptoms were closely associated with regulation of immune responses and gut microbiota. Bifidobacteria adolescentis interventions regulated the composition of gut microbiota, and thus indirectly altered SCFAs production. According to network analysis, immune responses were directly affected by the gut microbiome and SCFAs. Tregs, IFN-γ, and IL-10, related to inhibition of Th2 type immune responses, were positively associated with AF12, Bifidobacterium, and Anaerotruncus. Interestingly, there was a positive correlation between Tregs and butyric acid. Th2 type cytokines, such as IL-4 and IL-13 were negatively related to Bifidobacterium and Allobaculum. Additionally, the interaction between gut microbiota was important for AD amelioration. The increase in Lactobacillus and Bifidobacterium suppressed the relative abundance of Dorea, and AF12 and Anaerotruncus were contributed to a decrease in Pediococcus and Pseudomonas (negative correlation). Collectively, Bifidobacterium interventions were contributed to AD amelioration through inhibition of Th2 type immune responses, SCFAs production, and competition between gut microbiota.

Fig. 8
figure 8

Correlation networks and schematic diagram. a The interaction networks among clinical symptoms, immune indicators, SCFAs production, and differential taxa. The size of the circles stands for the contribution; red represents a positive correlation, green represents a negative correlation (P < 0.05, r > 0.3 or r < 0.3). b Schematic diagram for the alleviating effects of Bifidobacterium adolescentis on atopic dermatitis

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Discussion

This study evaluated the effects of six B. adolescentis strains on skin lesions, gut microbial profiles, and their immunomodulatory properties. The correlations between immune and pathological indices and specific gut microbiota were further analyzed, and changes in the functional gene in the metabolic pathways and SCFAs were explored.

AD is a common disorder with severe pruritus on the skin and significantly decreases the life quality index of patients. To explore the effect of B. adolescentis on AD, we established a DNFB-induced AD mouse model with a polarized Th2 immune response. Initial and final body weight of mice had no statistical difference within the group or between groups during the experiment period (Fig. S1). DNFB markedly destroyed the physical barrier of the ear and induced ear swelling (Fig. 1b). Additionally, in the skin lesions mast cells infiltration and collagen accumulation were increased and thus leading to dermal thickening and tissue damage (Fig. 2). Treatment with B. adolescentis Ad1, Ad3, and Ad6 reduced ear thickness, and the ear swelling was partially suppressed by these strains. It indicated that these strains had the potential to alleviate AD-like clinical symptoms. Therefore, we next evaluated the pathological changes in the dorsal skin of AD mice. Bifidobacteria adolescentis Ad1 and Ad3 also suppressed the increase in dermal thickening, and B. adolescentis Ad1 reduced mast cells infiltration and accumulation in skin lesions. The immune role of effective probiotics on alleviating AD-like symptoms would be explored because of the development of AD involved immune disorders.

Tregs have the ability to suppress Th2 immune responses by moving to peripheral tissues and inflammation draining lymph nodes during the allergic immune responses [24]. To investigate whether B. adolescentis increased differentiation of Tregs in the spleen, the flow cytometry analysis was performed. DNFB significantly reduced the CD4 + CD25 + Foxp3 + Treg population while the oral administration of B. adolescentis Ad1, Ad3, and Ad5 significantly promoted the ratio of Tregs in the spleen (Fig. 3b). Th1- and Th2-type immune responses are pivotal for allergic diseases. Therefore, the cytokines involving the balance and serum IgE levels were evaluated in AD-like skin lesions. Treatment with B. adolescentis decreased serum IgE levels except for A2 and Ad3, but B. adolescentis A1 and A2 suppressed IL-4, IL-5, and CCL22 levels versus DNFB group (Fig. 4a–e). However, B. adolescentis could not reduce the expression IL-13 levels except Ad5 and Ad6. Additionally, B. adolescentis A1, A2, and A3 significantly increased IFN-γ and IL-10 levels (Fig. 4f, g). These results indicated that B. adolescentis promoted Th1-type immune responses and suppressed Th2-type immune responses. In other words, treatment with B. adolescentis restored the balance related to Th1- and Th2-type immune responses. Besides, IL-10 also contributed to Th2 immune inhabitation and the increased IL-10 production might be associated with the migration of Tregs in the spleen [25, 26]. But studies are to be needed to explore the pathways involving the migration of Tregs among gastrointestinal, general, and skin immune system.

Studies have demonstrated that changes in gut microbial composition were associated with AD [27,28,29]. Gut dysbiosis is one of the causes to lead to AD. Our results showed that in the DNFB group Firmicutes decreased but Bacteroidetes prevailed and thus leading to a low F/B ratio (Fig. 5a, b). Treatment with B. adolescentis decreased the proportion of Bacteroidetes and increased Firmicutes and resulted in an increased F/B ratio and improved gut dysbiosis. To further explore the different taxa, we performed LEfSe analysis. We found that Pseudomonas, Sutterella, Enterococcus, Dorea, and Pediococcus were the core microbiome in the DNFB group (Fig. 6a, b). Sutterella has been reported to relate to the development of autism and is prevalent in children with autism and gastrointestinal dysfunction [30]. Dorea spp., major gas-producing bacteria [31], have been reported to increase in IBS patients [32]. Pseudomonas is widely distributed on the skin of humans and animals and leads to skin infection, urinary tract infection, and central nervous system infections [33]. The development of AD might be associated with the presence of these harmful bacteria in the DNFB group. However, the proportion of Lactobacillus was increased by B. adolescentis treatments versus the DNFB group (Fig. 6c). The increase in Lactobacillus was contributed to restoring the balance of gut microbiota under pathological conditions. These results demonstrated that B. adolescentis treatment altered gut microbial composition in AD mice, and affected the gut microenvironment.

Gut microbiota-produced SCFAs serve as energy substrates for epithelial cells and affect various physiological processes in the gut [34]. A study has reported that SCFAs regulate the function of the colonic Tregs and prevent colitis from developing in mice [35]. Treatment with B. adolescentis increased propionic acid and butyric acid concentrations but decreased isovaleric acid versus the DNFB group (Fig. 7a). The increased propionic acid was negatively related to clinical symptoms, CCL22, and Dorea (Fig. 8a), and indicated that propionic acid was contributed to the inhibition of these indicators, but it was associated with the increase in Lactobacillus. Butyric acid was also negatively correlated with clinical symptoms but positively correlated with IFN-γ. It indicated that butyric acid suppressed the clinical pathology and increased Th1 type immune responses. However, isovaleric acid was related to the increase of IgE, CCL22, and Dorea, and related to the aggravation of AD.

Gut microbial alteration affected not only SCFAs metabolism but the composition of the functional genes. Taking B. adolescentis Ad1 as an example based on a PCA analysis (Fig. 7b). 11 involved in KEGG pathways in the control and DNFB groups were identified, and treatments with B. adolescentis significantly influenced the functional gene composition (Fig. 7c). Pathways involved in fructose and mannose metabolism and fatty acid biosynthesis were upregulated by B. adolescentis Ad1 treatment (Fig. 7d), and this might be associated with the increase in propionic and butyric acids (Fig. 7a). Additionally, a study has reported that the reduction in genes involved in antigen processing and presentation and nucleotide-binding oligomerization domain-like receptor signaling was associated with impaired immune development in the six-month-old infants with AD versus healthy control subjects [36]. Our results showed that B. adolescentis Ad1 treatment also significantly upregulated both two pathways versus DNFB group (Fig. S3).

Besides the effect of SCFAs on clinical and immune indicators, gut microbiota was important in the network analysis (Fig. 8a). AF12 and Allobaculum were inversely related to IgE, CCL22, and IL-4, respectively, and Bifidobacterium and Lactobacillus were inversely related to IL-13 and IL-5, respectively. AF12 was positively related to Tregs, IL-10, and IFN-γ. Obviously, the increase in these genera restored the balance related to Th1 and Th2 type immune responses, and this was contributed to the inhibition of immune responses related to AD development. Additionally, the core microbiome in the DNFB group, such as Dorea was negatively related to Anaerotruncus, Ruminococcus, Lactobacillus, and Bifidobacterium and this revealed the competition between these genera in the gut. The competition in the gut helped the restoration of balance between gut microbiota in AD mice. Changes in gut microbiota caused by B. adolescentis treatment were associated with improved clinical symptoms, suppressed Th2 type responses, and increased SCFAs production according to network analysis. Therefore, the strain-specific ameliorating effect of B. adolescentis on AD manifestation closely related to the alteration of gut microbiota.

There are some limitations in this study, for instance, the communication between B. adolescentis and the gut immune system needs to be elucidated, and the metabolites except SCFAs contributing to immune regulation need to be identified as well as the related gut bacteria.

In summary, the results showed that the immune responses induced by DNFB were suppressed by B. adolescentis through promoting Tregs population in the spleen, restoring the balance between Th1- and Th2-type immune responses, and regulating the gut microbiota composition (Fig. 8b). Additionally, changes in SCFAs and functional gene profiles demonstrated the possible metabolism pathways in AD disease.