Bifidobacterium animalis subsp. lactis LKM512 Alleviates Inflammatory Bowel Disease in Larval Zebrafish by Reshaping Microbiota

Abstract

Inflammatory bowel disease (IBD) is a worldwide issue, and the increased incidence has brought a heavy burden to patients and society. Gut microbiota is involved in the pathogenesis of IBD, and targeting the microbiota, such as probiotics, has emerged as a potential therapy for the treatment of IBD. Here, the effect of Bifidobacterium animalis ssp. lactis LKM512 (LKM512), an anti-aging probiotic, on dextran sulfate sodium salt (DSS)-induced IBD in larval zebrafish was determined. Supplementation of LKM512 promoted the survival rate of the larvae, together with increased locomotor activities and body length. In addition, LKM512 treatment enhanced mucus secretion and alleviated intestinal injury, and these results were associated with the upregulation of mucin-related and downregulation of inflammatory markers. Moreover, LKM512 increased the diversity of the microbiota and ameliorated the dysbiosis by increasing the abundance of Bacteroidetes and Firmicutes and reducing the abundance of Proteobacteria. Specifically, the abundance of beneficial bacteria, including the short-chain fatty-acids (SCFAs)-producing genera Lachnospiraceae_NK4A136_group, Muribaculaceae, and Alloprevotella, was increased by LKM512, while the abundance of harmful genera, such as Pseudomonas, Halomonas, and Escherichia–Shigella, was reduced by LKM512. Consistent with these findings, the microbial functions related to metabolism were partly reversed by LKM512, and importantly, fermentation of short-chain fatty acids-related functions were enhanced by LKM512. Therefore, LKM512 might be one potential probiotic for the prevention and treatment of IBD, and further studies that clarify the mechanism of LKM512 would promote the application of LKM512.

INTRODUCTION

Inflammatory bowel disease (IBD),¹⁾ which consists of Crohn’s disease (CD) and ulcerative colitis (UC),²⁾ is a worldwide issue due to its complex clinical problems as well as difficulties in guaranteeing the efficacy of drugs.^3,4) With the increasing incidence of IBD,⁵⁾ more and more studies pay great attention to finding effective therapy for IBD.⁶⁾ Intestinal barrier integrity impairment and dysbiosis of microbiota are involved in the pathogenesis of IBD.⁷⁾ Intestinal epithelial cells are covered with a thick layer of mucus, including mucin secreted by goblet cells, immunoglobulin A (IgA) secreted by plasma cells, and antimicrobial peptides, which form a mucosal epithelial barrier that resists pathogenic microorganisms.^8,9) However, such a biological barrier is destroyed during the progression of IBD.¹⁰⁾ In addition, the composition of gut microbiota in IBD patients is altered, with an increased abundance of pathogenic bacterial species and a decreased abundance of beneficial bacteria.¹¹⁾ Strategies that target the intestinal barrier and microbiota have significantly increased in recent years.

Probiotics play a vital role in protecting the intestinal barrier by maintaining microbiota homeostasis and enhancing host immune function.^12,13) Studies have found the mutual support of Lactobacillus and Bifidobacterium to bacteria that suppress the growth of pathogenic bacteria by reducing the pH in the intestinal environment,¹⁴⁾ and thereby used as a therapeutic approach or adjuvant therapy for various diseases.^15,16) Bfidobacterium longum is also found to upregulate the transforming growth factor β (TGF-β) signaling of paneth cells to repair the damaged barrier function and restore the proliferation of intestinal stem cells in mice.^17,18) In addition, emerging evidence has shown that certain probiotic bacteria could prevent or minimize intestinal inflammation in animal models, and recent clinical evidence also supports using probiotics in IBD patients.¹⁹⁾ We have recently found that Bifidobacterium longum BL986 (BL986) and Lactobacillus casei LC122 (LC122) exerted different effects on IBD in zebrafish, with BL986 being more potent in the dextran sulfate sodium salt (DSS)-induced UC model and LC122 exerted better protection against 2,4,6-trinitro-benzenesulfonic acid (TNBS)-induced CD.²⁰⁾ However, more probiotic strains with potent IBD-amelioration effects are still needed.

Bifidobacterium animalis ssp. lactis LKM512 (LKM512), an anti-aging probiotic, demonstrates potent acid tolerance and the ability to adhere to intestinal mucin.²¹⁾ The administration of LKM512 exerts an anti-inflammatory effect, as well as reshaping the composition of the intestinal microbiota of the elderly.^22,23) In our previous study, we found that the supplementation of LKM512 attenuated obesity-associated inflammation and insulin resistance by modifying microbiota in high-fat diet-induced obese mice.²⁴⁾ However, the potential effects of this probiotic on IBD remain unknown. To that end, the impact of LKM512 on IBD was investigated in the DSS-induced UC model in larval zebrafish.

MATERIALS AND METHODS

Animals

Wild-type AB zebrafish (Danio rerio) were purchased from Shanghai FishBio Co., Ltd. (Shanghai, China) and maintained under standard laboratory conditions with the temperature at 28 ± 1 °C and a 14 : 10 h light/dark cycle according to the zebrafish breeding protocol. The pH and hardness of water were 7.2–7.6 and 9.9–61.0 mg CaCO₃/L, respectively. Embryos were collected after spawning and soaked in an E3 medium until hatched, as described previously.²⁰⁾ Larvae at 3 d post-fertilization (dpf) were used for further experiments.

Construction of IBD Zebrafish Model

Dextran sodium sulfate (DSS) is commonly used to induce a UC-like IBD zebrafish model.²⁵⁾ In the present study, 3 dpf larval zebrafish were incubated in E3 medium with 0.5% DSS (Yeasen Biotechnology, Shanghai, China) for 4 d to construct the IBD model. The Bifidobacterium animalis ssp. lactis LKM512 (LKM512, Meito Sangyo Co., Ltd., Tokyo, Japan) was simultaneously added in the E3 medium from 3 to 7 dpf with a final concentration of 1 × 10⁴ colony forming unit (CFU)/mL and 1 × 10⁵ CFU/mL, respectively. The DSS- and LKM512-containing medium was changed every 24 h. The mortality was recorded daily, and the locomotor activities were monitored at 5 and 7 dpf. All experiments were approved by the laboratory animals ethical committee of the Zhejiang University of Technology and followed NIH guide for laboratory animals (NIH Publication No. 85-23, revised 1996) for the care and use of animals.

Histological Analysis

Larval zebrafish were fixed in 4% paraformaldehyde, rinsed with acid alcohol, and stained with 0.01% alcian blue (Sangon, Shanghai, China, w/v, in 80% ethanol and 20% glacial acetic acid). Hematoxylin–eosin (H&E) staining was performed after the larvae were soaked in Bouin’s fluid (Acmec Biochemical, Shanghai, China), gradient dehydrated, fixed, and paraffin-embedded, as described previously.²⁰⁾

RNA Extraction, Reverse Transcription, and Quantitative (q)PCR Analysis

Fifty larval zebrafish from the same beaker were mixed as one sample for total RNA extraction using Biozol RNA Reagent (Biomiga, Hangzhou, China). Then, a total of 1000 ng RNA from each sample was used to synthesize cDNA by a SynScript III RT SuperMix (Tsingke Biotechnology Co., Ltd., Beijing, China). The real-time qPCR was performed using the 2 × T5 Fast qPCR Mix (Tsingke Biotechnology Co., Ltd.) on a CFX Connect Optics Module (Bio-rad, U.S.A.). The following PCR protocol was used: denaturation for 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C for renaturation and elongation. The sequences of primers used in this study are listed in Table 1.

Table 1. List of Primer Sequences Used for RT-PCR Analysis

Target gene	Sequence of the primers (5′ to 3′)
cldn 11	Forward, 5′-CCACGATGGAGTTACCAGCTA-3′
	Reverse, 5′-TGTGTCTGTGTGAGTTTGAGTGTT-3′
cldn 2	Forward, 5′-TATCGTTGATTCCCGTCGCC-3′
	Reverse, 5′-TCATCGCAACAGGATGCACT-3′
muc2.2	Forward, 5′-ACACGCTCAAGTAATCGCACAGTC-3′
	Reverse, 5′-TCAGCGAGTGTTTGGCTCACTT-3′
muc2.1	Forward, 5′-CAACATCGATGGCTGCTTCTG-3′
	Reverse, 5′-CTGACAGTAACATTCTTCCTCGC-3′
il1β	Forward, 5′-ATCAAACCCCAATCCACAGAGT-3′
	Reverse, 5′-GGCACTGAAGACACCACGTT-3′
il-6	Forward, 5′-TCAACTTCTCCAGCGTGATG-3′
	Reverse, 5′-TAAAGCACTCCACAACCCCA-3′
tnfα	Forward, 5′-GCGCTTTTCTGAATCCTACG-3′
	Reverse, 5′-TGCCCAGTCTGTCTCCTTCT-3′
mmp9	Forward, 5′-GCTCAACCACCGCAGACTAT-3′
	Reverse, 5′-GTGCTTCATTGCTGTTCCCG-3′
p65	Forward, 5′-AAGATCTGCCGAGTGAACCG-3′
	Reverse, 5′-GCCTGGTCCCGTGAAATACA-3′
myd88	Forward, 5′-CAGTGGTGGACAGTTGTGGAC-3′
	Reverse, 5′-GAAAGCATCAAAGGTCTCAGGTG-3′
irf8	Forward, 5′-CACTGACATGGACTGCTCAC-3′
	Reverse, 5′-CATTCTTGCACTGAAGGCATG-3′
ef1a	Forward, 5′- ACCTACCCTCCTCTTGGTCG-3′
	Reverse, 5′-GGAACGGTGTGATTGAGGGAA-3′

DNA Extraction, PCR Amplification, and 16S Ribosomal RNA (rRNA) Sequencing

The genomic DNA (gDNA) was extracted from 100 larval zebrafish with the Ezup Column Soil DNA Purification Kit (Sangon, Shanghai, China) following the manufacturer’s instructions. Then, the gDNA samples were amplified using specific primers (Forward primer: 5′-GTGYCAGCMGCCGCGGTA-3′; Reverse primer: 5′-CCCCGYCAATTCMTTTRAGT-3′) targeting the V3 and V4 regions of the bacterial 16S rRNA gene. The PCR protocol was carried out as follows: 95 °C for 5 min, 95 °C for 15 s, 50 °C for 15 s, and 72 °C for 15 s, repeated for 40 cycles, followed by 72 °C for 5 min. The DNA libraries were validated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, U.S.A.), and quantified using PicoGreen dsDNA quantitation Reagent (Yeasen, Shanghai, China) by UV spectroscopy. The Illumina Hiseq sequencing was conducted at Novogene (Tianjin, China).

Microbiota Data Analysis

Raw sequence data were demultiplexed using the demux plugin, followed by primers cutting with the cutadapt plugin. Sequences were then quality filtered, denoised, merged, and chimera removed using the DADA2 plugin. As described previously, the microbiota composition was mainly determined by QIIME2 bioinformatic analysis and R packages (v3.2.0).²⁶⁾ Taxonomic classifications were conducted using the Greengenes database,²⁶⁾ and the composition of microbial communities was visualized using linear discriminant analysis effect size (LEfSe).²⁷⁾ Microbial functional profiling was predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt).²⁶⁾

Statistical Analysis

All data were shown as the mean ± standard error of the mean. Differences between the mean values were assessed using a one-way ANOVA followed by the Tukey–Kramer test. p < 0.05 was considered statistically significant. False discovery rate (FDR) multiple testing corrections were applied to the resulting p-values with a less strict significance level (FDR q-value of 0.1) to ensure data accuracy, as described previously.²⁸⁾

RESULTS

LKM512 Promoted the Survival Rate and Growth of Larval Zebrafish in the DSS-Induced IBD Model

DSS treatment caused a marked decrease in the survival rate of the larval zebrafish from 5 dpf, and resulted in a 15.8% reduction at the 7 dpf (Fig. 1A). LKM512 administration at both doses significantly increased the survival rate of the larvae, leading the final survival rate to approximately 94% at the end of treatment (Fig. 1A). The survival rate-promoting effect of LKM512 was similar in both doses, and the subsequent experiments were mainly performed using 1 × 10⁵ CFU/mL. The locomotor activity was also attenuated by DSS treatment during the light/dark stimulation, especially in the dark phase, and tended to be enhanced by LKM512 (Fig. 1B). In addition, the body length of the larvae was markedly reduced at the 5 dpf, which was not affected by LKM512 (Fig. 1C). However, the body length was increased by LKM512 at the 7 dpf (Fig. 1D).

Fig. 1. LKM512 Promoted the Survival Rate and Growth of Larval Zebrafish

(A) The survival rate of different groups after DSS and LKM512 treatment from 4 to 7 dpf, n = 6, 120 larvae were maintained in a 6-well plate (20 larvae per well), and the survival rate of each well was calculated. (B) The activity counts during the light/dark stimulation, n = 24 for each group. (C, D) The body axis length of each group at 5 dpf (C) and 7 dpf (D), n = 15 for each group.

LKM512 Ameliorated Intestinal Damage and Inflammation in DSS-Treated Larval Zebrafish

Alcian blue staining, as an indicator of mucus protein, revealed that DSS treatment caused a great decrease in mucin content in the intestinal tract of larval zebrafish, which was increased by the LKM512 treatment (Fig. 2A). Similarly, H&E staining indicated that DSS caused severe intestinal injury, as characterized by the damage to the mucosal layer and loss of typical intestine folding and the increase of cell shedding (Fig. 2B). LKM512 treatment yielded protective effects on the intestinal barrier function, with notably intact crypts and less inflammatory cell infiltration (Fig. 2B). Consistent with these findings, qPCR results found that the mRNA expression of mucin-related markers, such as muc2.1, was significantly downregulated in DSS-treated larval zebrafish, and markedly upregulated by LKM512 (Fig. 2C). However, no significant changes were found in the expression of muc2.2 and tight junction-related genes by DSS or LKM512 treatment, such as claudin 11 (cldn11) and cldn2 (Fig. 2D). Moreover, the expression of inflammatory markers, including transcription factor p65, interleukin-6 (il-6), myeloid differentiation primary response protein 88 (myd88), matrix metallopeptidase 9 (mmp9), interferon regulatory factor 8 (irf8), and il-1β, were all upregulated in DSS-treated group, and the expression of p65, il-6, mmp9, and il-1β were significantly downregulated by treating with LKM512 (Fig. 2E).

Fig. 2. LKM512 Ameliorated Intestinal Damage and Inflammation in Larvae Zebrafish

(A) Alcian blue staining of the larvae, black arrow indicated the alcian blue-positive mucin. (B) H&E staining of intestinal cross-sections of larvae at 7 dpf under different magnifications, black arrow indicated the typical intestine folding or the increase of cell shedding. (C) qPCR analysis of mucin-related genes in the larvae, 50 larvae were mixed as one sample, n = 6–8 for each group. (D) qPCR analysis of tight junction-related genes in the larvae, 50 larvae were mixed as one sample, n = 6–8 for each group. (E) qPCR analysis of inflammatory genes in the larvae, 50 larvae were mixed as one sample, n = 6–8 for each group.

LKM512 Reshaped the Microbial Composition and Function in IBD Zebrafish

Next, the effect of LKM512 on the composition and function of microbiota was determined by 16S rRNA sequencing. The α-diversity of microbiota, as indicated by the Margalef and Shannon index, decreased in DSS-treated larvae and was significantly increased by LKM512 treatment (Fig. 3A). Principal component analysis (PCA) of the β-diversity revealed clear separation in the microbial community structures of three groups (Fig. 3B). The overall microbial composition at the phylum level was markedly altered by either DSS or LKM512 (Fig. 3C). Specifically, the relative abundance of Bacteroidetes and Firmicutes decreased by 28.9% and 44.2% in DSS-treated group, respectively, which were increased by LKM512 by 2.4- and 2.1-fold, respectively (Fig. 3C). While the abundance of Proteobacteria was increased by 15.8% by DSS treatment, and reduced by 26.8% in LKM512-treated larvae (Fig. 3C). At the genus level, DSS and LKM512 treatment also resulted in a marked difference in the abundance of top 40 abundant species, with 18 genera increased, including Bacteroides, Alistipes, Oscillibacter, Lachnospiraceae_NK4A136_group, Oscillospiraceae, Lachnospiraceae, Muribaculaceae, Alloprevotella, and others, and 22 genera decreased, such as Pseudomonas, Halomonas, Bradyrhizobium, Mycobacterium, Caulobacterales, Corynebacterium, Escherichia–Shigella, and others, after the administration of LKM512 (Fig. 3D). Consistent with these findings, the LEfSe analysis also revealed obvious different signature of the taxa in DSS- and LKM512-treated group (Fig. 4A). For example, feature taxa in DSS-treated group were mainly belong to the Proteobacteria, and those in LKM512-treated group were mostly belong to Bacteroidetes and Firmicutes (Fig. 4A).

Fig. 3. LKM512 Altered the Composition of Microbiota in IBD Zebrafish

(A) Margalef index and Shannon index. (B) β-Diversity of microbiota in different groups. (C) Relative abundance of microbiota at the phylum level. (D) Heatmap of top 40 genera in the larvae. 100 larvae were mixed as one sample, n = 6 for each group.

Fig. 4. LKM512 Reshaped the Functional Profiling of Microbial Communities

(A) Cladograms generated from LefSe analysis showing the microbial clades with the great differences in abundance in microbiota from Con (blue), DSS (red), or LKM512 (green) group. (B) Functional profiles derived from PICRUSt analysis between Con and DSS, and DSS and LKM512 group. n = 6, star means Q value <0.1.

The functional profile of the microbial community was predicted by PICRUSt analysis and found significant differences between the control and DSS groups and the DSS and LKM512-treated groups (Fig. 4B). In detail, the functions related to nucleoside and nucleotide biosynthesis and degradation, carbohydrate biosynthesis and degradation, secondary metabolite biosynthesis, and other functions slightly or greatly reversed by LKM512 treatment (Fig. 4B). However, the function associated with aromatic compound biosynthesis and degradation, cofactor, prosthetic group, electron carrier, and vitamin biosynthesis, amino acid biosynthesis and degradation were not significantly affected by LKM512 (Fig. 4B). Importantly, fermentation of short-chain fatty acids-related functions, such as pyruvate fermentation to butanoate and superpathway of Clostridium acetobutylicum acidogenic fermentation, decreased significantly in DSS group, which were enhanced by LKM512 (Fig. 4B).

DISCUSSION

The pathogenesis of IBD is complicated and remains not fully elucidated,²⁹⁾ and it is well-known that the IBD and the subsequent epithelial barrier damage lead to dysregulated interactions between the microbiota and host.³⁰⁾ A growing body of studies have highlighted the beneficial effect of probiotics on restoring the balance of gut microbiota and ameliorating the symptoms of IBD.³¹⁾ For example, we have recently found that Bifidobacterium longum BL986 (BL986) and Lactobacillus casei LC122 (LC122) alleviated IBD in zebrafish in a disease subtype- and age-dependent manner, and the mechanism of these two probiotics was also acting differently.²⁰⁾ Here, we also found that the supplementation of LKM512 protected the intestinal barrier and altered the composition and function of microbiota in larval zebrafish. Though the effect of LKM512 was similar to those of BL986 in the DSS-induced UC model, as both probiotic strains belong to the Bifidobacterium family, the mechanism might still be different. For instance, the effect of LKM512 on microbiota was different from that of BL986 in the larvae, with a stronger impact on the abundance of short-chain fatty acids (SCFAs)-producing genera, such as Lachnospiraceae_NK4A136_group,³²⁾ Muribaculaceae,²⁴⁾ Alloprevotella.²⁴⁾ The increased abundance of Lachnospiraceae_NK4A136_group was also consistent with our finding in obese mice.²⁶⁾ Therefore, the beneficial effect of LKM512 might be associated with reshaping the microbial composition.

The change of composition in microbiota is deemed to cause a marked alteration of the functional profile, which was also verified by LKM512 treatment in IBD zebrafish. Interestingly, the effect of LKM512 on the microbial function related to carbohydrate and amino acid metabolism was mild in the present study, which was different from the findings of the effect of LKM512 on metabolic disease,²⁶⁾ and also our previous study of BL986 on IBD zebrafish.²⁰⁾ Here, we found that LKM512 was more prominent in affecting the functions related to SCFAs fermentation, especially the butyrate. Importantly, microbiota-derived butyrate is associated with the induction and differentiation of regulatory T cells (Tregs),³³⁾ and the induction of Tregs is a central mechanism of host-microbiota homeostasis.³⁴⁾ Moreover, activation of Tregs contributes to the amelioration of colonic inflammation.^35,36) Therefore, the effect of LKM512 on colonic inflammation might be mediated by microbial butyrate-induced activation of Tregs. Further studies that determine the SCFAs level and other metabolites and the correlation analysis would provide more details about the mechanism of how LKM512 reshapes the microbial composition to alleviate IBD.

Intestinal barrier dysfunction, especially the breakdown of the mucosal barrier and increased inflammatory cytokines production, is another characteristic feature of IBD.³⁷⁾ Strategies that target the barrier function, such as increasing mucin secretion, enhancing the tight junction of the epithelial cells, and reducing the inflammatory mediators, have been greatly utilized in the prevention and treatment of barrier dysfunction-related diseases.³⁸⁾ In the present study, we found that LKM512 significantly upregulated the expression of mucin-related genes and downregulated the expression of inflammatory genes, thereby protecting the intestinal barrier function, as evidenced by the morphology change in the gut of the larvae. On the other hand, we have previously found that the administration of LKM512 increased polyamine, mainly spermidine concentration, in the gut of mice.²⁶⁾ Spermidine is found to protect intestinal barrier function and alleviate inflammation in IBD mice.^32,39) Therefore, the protective role of LKM512 in IBD might be partly mediated by enhancing polyamine metabolism. Further studies that determine the polyamine levels and clarify how LKM512 protects the intestinal barrier function are needed.

On the other hand, there was still a possibility that bacterial cells reduced the effective concentration of DSS. However, the effect of probiotics on the DSS-induced IBD model in zebrafish was also determined by our team²⁰⁾ and others using similar exposure methods.^40–42) In addition, the DSS- and bacteria-containing medium was changed every 24 h during the exposure to minimize the influence of effective DSS concentration on the experiment results in the present study. Thereby, the effect of LKM512 on IBD was mainly attributed to the probiotic itself but not the reduction of DSS toxicity. Although the current study did not investigate the effect of LKM512 alone on control larvae, while it was speculated that LKM512 might also exert some improvements in the control zebrafish, such as promoting the growth and development and enhancing the immunity and intestinal barrier function of the larvae, similar to the findings of our previous probiotic study in zebrafish.²⁰⁾ Further studies are required to fully understand the mechanism of LKM512 on gut health.

In summary, LKM512 treatment promoted the survival rate, locomotor activity, and body length of larval IBD zebrafish, and these effects were associated with improved intestinal barrier function and altered microbial composition and function. Further studies that focus on the mechanism of LKM512 on IBD will provide more details about the effect of LKM512, which would also lay the theoretical basis for the application of LKM512 in the prevention and treatment of IBD in the future.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFD1700401, 2017YFD0200503).

Conflict of Interest

The authors declare no conflict of interest.

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