Bile salt hydrolase of Lactiplantibacillus plantarum plays important roles in amelioration of DSS-induced colitis

Summary Bile salt hydrolases are thought to be the gatekeepers of bile acid metabolism. To study the role of BSH in colitis, we investigated the ameliorative effects of different BSH-knockout strains of Lactiplantibacillus plantarum AR113. The results showed that L. plantarum Δbsh 1 and Δbsh 3 treatments did not improve body weight and alleviate the hyperactivated myeloperoxidase activity to the DSS group. However, the findings for L. plantarum AR113, L. plantarum Δbsh 2 and Δbsh 4 treatments were completely opposite. The double and triple bsh knockout strains further confirmed that BSH 1 and BSH 3 are critical for the ameliorative effects of L. plantarum AR113. In addition, L. plantarum Δbsh 1 and Δbsh 3 did not significantly inhibit the increase in pro-inflammatory cytokines or the decrease in an anti-inflammatory cytokine. These results suggest that BSH 1 and BSH 3 in L. plantarum play important roles in alleviating enteritis symptoms.


INTRODUCTION
Inflammatory bowel diseases (IBDs) are mainly divided into ulcerative colitis (UC) and Crohn's disease, the typical symptoms of which include abdominal pain, diarrhea, blood in the stool, loss of appetite, fatigue, and weight loss. 1 The incidence and prevalence of IBD have increased markedly in recent years, making IBD a global public health problem, which has attracted worldwide attention. Although the exact cause of IBD remains largely unknown, several studies have suggested that it involves a complex interaction between genetic, microbial, and immune factors. [1][2][3][4][5] Recent research has revealed that of these factors, the composition of the intestinal microbiota is especially important in IBD pathogenesis. [3][4][5] Some recent studies have suggested that the intestinal microbiota affects IBD pathogenesis through metabolites such as short-chain fatty acids (SCFAs) and bile acids (BAs). [6][7][8][9][10][11][12] SCFAs metabolized by intestinal bacteria have been shown to ameliorate the typical symptoms in animal models of IBD and have been associated with a reduced risk of IBD. [7][8][9][10] In addition, some recent studies have found that changes in BAs and BA metabolism are also linked to IBD. [10][11][12][13] Studies have found that although the levels of primary BAs and conjugated BAs (CBAs) are augmented in the fecal samples of healthy individuals, the levels of secondary BAs are decreased in the fecal samples of IBD patients, however, it is not clear why IBD patients manifest these changes and what role these changes play in IBD pathogenesis. 11,12 Intestinal microorganisms can promote the conversion of primary BAs to secondary BAs, thus changing the composition of BAs in the body. As bile salt hydrolases (BSHs) can cleave the amide bond in conjugated BAs to increase the concentration of deconjugated BAs, which can subsequently undergo a variety of transformations to generate secondary BAs, they are thought to be the gatekeepers of BA metabolism and host microbiome crosstalk in the gastrointestinal tract. 14,15 Labbe et al. 13 found a significant reduction in the abundance of the Firmicutes-derived BSH (bsh) gene in IBD patients relative to healthy controls, using large available datasets containing metagenomic information from IBD patients. Studies have also reported a significant negative correlation between the relative abundance of bacterial bsh genes and the retinoic acid receptor-related orphan receptor gamma (RORC) gene, which is related to IBD. 11,16 Using chemoproteomic approaches, Parasar et al. 17 identified altered BSH activities in a murine model of IBD. In silico analysis showed that the relative abundance of BSH in the gut microbiota was markedly lower in IBD patients than in healthy individuals, and that this reduction was most evident in Firmicutes from the patients. 17 Although an association of BSH in the gut microbiota with IBD has been suggested, further research is warranted to provide the evidence. Current treatment strategies for IBD focus on reducing the inflammatory burden in patients with active disease and maintaining remission in those with inactive disease. 18,19 However, they are associated with a wide range of possible severe side effects, and some of these treatments are costly. 18-20 As a result, probiotics are attracting increasing research interest as a safer way of ameliorating disease activity. 20 Several studies have found that some probiotics can ameliorate or prevent IBD. 20-24 Although the mechanisms of the ameliorative effects of probiotics on IBD are not well defined, many mechanisms have been proposed to explain this action, including antagonism to pathogenic bacteria, modulation of gut microbiota, production of nutrients, and enhancement of anti-inflammatory cytokine levels. 20-24 A meta-analysis showed that probiotic VSL#3 is effective in inducing remission in active UC and is equivalent to 5-aminosalicylic acid (5-ASA) in preventing UC relapse. 20 Wang et al. 21 found that Lactiplantibacillus plantarum ZS2058 was an efficient producer of conjugated linoleic acid (CLA) in vitro and that it ameliorated dextran sulfate sodium (DSS)-induced acute colitis by producing CLA locally in mice. Clostridium butyricum producing high levels of SCFAs was revealed to alleviate epithelial damage in rats with DSS-induced colitis. 22 Ke et al. 23 demonstrated that fucose ameliorated intestinal inflammation by regulating the crosstalk between BAs and the gut microbiota in DSS-treated mice. However, few studies have evaluated the role of BSH in the ameliorative effect of probiotics on IBD. The family Lactobacillaceae is rich in BSHs. A total of 551 BSHs from 107 Lactobacillaceae species were identified from 451 genomes of 158 Lactobacillaceae species. 24 Through metagenomic analyses, Jones et al. 15 demonstrated that BSH activity is a conserved microbial adaptation to the human gut environment with a high level of redundancy in this ecosystem. Therefore, it is necessary to investigate whether probiotics exert ameliorative effects on IBD through BSHs. Our previous work found that L. plantarum AR113 was more effective than other probiotic strains in alleviating epithelial damage, improving colon length, and maintaining the epithelial barrier integrity after DSS treatment. 25 We also found that L. plantarum AR113 had the highest BSH activity among the 10 lactic acid bacterial strains tested with the plate assay. Furthermore, using in silico molecular docking, heterologous expression, and knockout experiments, we verified that the bsh 1 and bsh 3 genes were responsible for most of the BSH activity in L. plantarum AR113. 26,27 To explore the roles of BSHs in IBD, here we investigated the ameliorative effects of BSH-knockout strains of L. plantarum AR113 on the disease severity of mice with DSS-induced colitis.

BSH improves colitis symptoms
To explore the roles of BSHs in IBD, the effects of orally administrated BSH-knockout strains on the severity of DSS-induced colitis in mice were evaluated based on body weight loss, DAI, colon length, MPO activity, and histopathology. Mice were fed according to the protocol outlined in Figure 1.
The DSS group showed severe body weight loss from day 4 and demonstrated a final weight loss of 15% relative to the Control group. In the L. plantarum AR113 group, the DSS-treated mice showed evident recovery of body weight, with their weights being almost the same as those in the 5-ASA group and even approaching the Control group (Figure 2A). Compared with L. plantarum AR113, L. plantarum Dbsh 1 and  iScience Article (6.03 G 0.17) groups did not differ significantly from one another and were significantly higher than that of the L. plantarum AR113 group (4.20 G 0.40, p< 0.05, Figures 2D and S1D).
The colon lengths of mice in different groups were measured after euthanizing the mice ( Figure 2E). Compared with the Control group (6.82 G 0.31), the DSS group (5.90 G 0.53) showed significantly decreased colon length. The L. plantarum AR113 treatment group (7.1 G 0.18) effectively prevented the DSS-induced shortening of the colon and recovered the colon length to that observed in the Control and 5-ASA groups. The colon lengths in the L. plantarum Dbsh 1, L. plantarum Dbsh 3, L. plantarum Dbsh 13, L. plantarum Dbsh 132, and L. plantarum Dbsh 134 groups were significantly reduced relative to the L. plantarum AR113 treatment group and were not significantly different from one another. Figure 2F shows that the MPO activity in the DSS group (1.24 G 0.04 U/g) was significantly higher than that in the Control group (0.85 G 0.04 U/g), indicating that the colons of DSS-treated mice had significant neutrophil infiltration and severe inflammation.

Effect of BSHs on the inflammatory cytokine expression
Changes in the levels of pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and the anti-inflammatory cytokine IL-10 in mouse colon tissues were measured to determine the anti-inflammatory effect of bsh. The sequences of primers are listed in Table 1. As shown in Figure 4, compared with the Control group, the DSS group demonstrated a significant increase in the transcript levels of the colonic pro-inflammatory cytokines TNF-a, IL-1b, and IL-6, but a significant decrease in the transcript level of the anti-inflammatory cytokine IL-10. Similar to the 5-ASA group, the L. plantarum AR113 group showed significantly downregulated expression of the pro-inflammatory cytokines and upregulated expression of the anti-inflammatory cytokine relative to the DSS group. Similar to L. plantarum AR113, L. plantarum Dbsh 2 and L. plantarum Dbsh 4 also downregulated the expression of the pro-inflammatory cytokines and upregulated the expression of the anti-inflammatory cytokine. This indicates that BSH 2 and BSH 4 do not affect the inflammatory cytokine expression. In contrast to the L. plantarum AR113 group, the L. plantarum Dbsh 1 and L. plantarum Dbsh 3 groups showed no significant difference in the expression of most of inflammatory cytokines relative to the DSS group. Furthermore, the cytokine expressions in the L. plantarum Dbsh 13, L. plantarum Dbsh 132, and L. plantarum Dbsh 134 groups were not significantly different from one another or from those in the DSS group. These findings suggest that BSH 1 and BSH 3 in L. plantarum AR113 play crucial roles in regulating the inflammatory cytokine expression in the colon tissues of DSS-treated mice.

Effect of BSH on the expression of tight junction (TJ)-related genes
The relative expression of MUC2 secreted by the mouse colon tissues was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The primers information are listed in Table 1.The mRNA expression of MUC2 was significantly downregulated in the DSS group compared with the Control group ( Figure 5A). Compared with the DSS group, the 5-ASA, L. plantarum AR113, L. plantarum Dbsh 2, and  Effect of BSH on the total bile acid and the BA-specific receptors expression BA metabolism of IBD patients is damaged because of the impaired microbial enzyme activity, which result in the abnormal change of total bile acid (TBA). The ameliorative effect of BSH on IBD was further demonstrated by measuring the TBA in feces. The DSS group showed significantly higher of TBA compared with the Control group, the L. plantarum AR113 reduce effectively TBA to normal level ( Figure 6A). However, the remission effect of L. plantarum Dbsh1 and L. plantarum Dbsh134 was significantly worse than that of the L. plantarum AR113 ( Figure 6A). To determine if the change of TBA is because of the strains number, red fluorescent protein (RFP) gene was successfully inserted into the L. plantarum AR113 and knockout strains genomes using CRISPR technology. There is no change of fluorescence intensity of L. plantarum AR113, L. plantarum Dbsh1 and L. plantarum Dbsh134 in mice feces on days 8, 10 and 12, which shows that there was no significant difference in the number of strains ( Figure 6B). These findings show that the mutant strains survive in the mice to similar levels as the wild strain.
We further investigated the effect of BSH on the BA-specific receptors expression. Compared with the Control group, the expression of fgfr4, fgf15, shp and fxr are downregulated in DSS group significantly ( Figures 7A-7D), but the expression of cyp7a1 is significantly increased ( Figure 7E). L. plantarum AR113 treatments, in contrast, effectively alleviated the DSS-induced downregulated of fgfr4, fgf15, shp and upregulated of cyp7a1, even approaching to the normal levels ( Figure 7). L. plantarum Dbsh 1 and L. plantarum Dbsh 134 groups have a similar regulatory effect, but their effect was significantly worse than that of L. plantarum AR113 and was closer to that in the DSS group (Figure 7). These results show that L. plantarum can regulate BA-specific receptors expression BA receptor expression through BSH.

DISCUSSION
In our study, L. plantarum AR113 relieved the symptoms of DSS-induced colitis in mice by reducing DAI values, inhibiting the hyperactivated MPO activity, and increasing the colon length, but the bsh 1 or bsh 3 knockout strains did not. To the best of our knowledge, this is the first study to demonstrate probiotics can exert ameliorative effects on IBD through BSHs. BSH is a crucial enzyme that catalyzes an essential gateway reaction in BA metabolism. Our research also found that L. plantarum can affect total bile acid by BSH. This suggests that probiotics may influence the occurrence of inflammation by affecting BA iScience Article metabolism. Many studies have suggested that BSH activity and subsequent BA modification could significantly impact the pathophysiology of metabolic diseases, such as obesity, diabetes, and atherosclerosis, through perturbations of the BA pool. 28,29 Therefore, it is worth further studying the effect of probiotics on these metabolic diseases by changing BA and establish the corresponding relationship between BSH activity and prebiotic function.
In addition, we demonstrated the role of BSH in alleviating inflammation, providing a basis for rational and accurate screening of probiotics. To our best knowledge, few studies have examined the active component of probiotic that alleviate inflammation by constructing knockout strains. SCFAs are metabolized by gut bacteria have been shown to ameliorate inflammatory. 10 Although the exact source for the action of SCFA are still not clear, some probiotics that can reduce inflammation do not produce SCFAs. The exact function of SCFAs produced by probiotics has not been studied by knocking out SCFAs synthetic genes. We found that BSH is the active component in alleviating inflammation through single, double, and triple bsh knockout strains. So BSH activity can be included in the criteria for the anti-inflammatory probiotic strain selection.
In our study, L. plantarum AR113 treatment significantly downregulated the expression of TNF-a, IL-6, and IL-1b and upregulated that of IL-10, however, L. plantarum Dbsh 1 and L. plantarum Dbsh 3 treatments did not affect the inflammatory cytokine expression. This indicated that BSH are involved in the regulation of these factors. Some studies have demonstrated that BAs inhibit the secretion of TNF-a, IL-1b, and IL-6 in macrophages, and that this downregulation is mediated by the BA-specific membrane receptor TGR5. 30 Some studies also found that BA-dependent farsenoid X receptor (FXR) activation appears to limit mucosal inflammatory responses via reducing pro-inflammatory cytokine (e.g., IL-1b, IL-6). 31 In this study, L. plantarum AR113 significantly upregulated of FXR expression, whereas L. plantarum Dbsh1 and L. plantarum Dbsh134 did not. This suggests L. plantarum AR113 may modulate inflammatory balance by regulating bile salts. Parasar et al. 17 identified altered BSH activities in a murine model of IBD, which led to changes in BA metabolism that could impact host metabolism and immunity. They also reported that Firmicutes-derived BSH was significantly reduced in IBD patients relative to healthy individuals. This suggests from another aspect that oral administration of L. plantarum can regulate BA-dependent immune responses to alleviate DSS-induced colitis through the BSH activity.

Limitations of the study
This study focuses on the role of BSH in studying the ameliorative effects of BSH in enterohepatic circulation and the ameliorative effects of BSH on the disease severity of mice with DSS-induced colitis, whereas the mechanism of BSH alleviating the colitis needs more detailed research. It is well known that a balanced and intact intestinal environment is closely related to intestinal health and disease treatment; it would thus iScience Article be interesting to investigate the changes of intestinal flora community, abundance changes and potential mechanisms.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  The data reported in this paper will be shared upon request to the lead corresponding author (1015wanggq@163.com).
This paper dose not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Strains and culture conditions L. plantarum AR113 and its seven bshmutant strains were obtained from Shanghai Engineering Research Center of Food Microbiology, University of Shanghai for Science and Technology (Shanghai, China). 32 All strains were anaerobically cultured in deMan, Rogosa, and Sharpe (MRS) medium at 37 C for 16h before centrifugation (3,000 3 g for 3minat 4 C). The cell pellets were washed twice with phosphate buffer solution (pH 7.4) and then resuspended at a density of approximately 5 3 10 9 CFU/mL, which was determined by colony counting on MRS plates, for the following experiments. Each mouse was orally administered with 1 x10 9 CFU of the strains in 0.2mL (5 3 10 9 CFU/mL) by oral gavage daily from day 5 to day 12 of the experiment. iScience Article allowed at least 1 week to adapt to the experimental environment before conducting the following experiments. The experimental procedures were performed in accordance with the institutional and governmental regulations on the ethical use of experimental animals.

Animals and experimental design
The mice were randomly divided into 11 groups of eight ( Figure 1). The Control group was given free access to sterile water and fed a normal diet for the whole experimental period (12 days). The DSS group was given free access to 2.5% DSS-containing drinking water for the first 7 days and then fed normal diet and normal water for the next 5 days of the experiment. The DSS-treated mouse groups of L. plantarum AR113 and its seven bshmutant derivatives were administered the corresponding strains (0.2 mL containing 5 3 10 9 CFU/mL) once a day by gavage from day 5 to day 12. The 5-ASA group of DSS-treated mice was administered 5-ASA (0.2mL) once a day by gavage from day 5 to day 12.

Assessment of MPO activity
The colonic MPO activity in each group of mice was determined using an MPO test kit (Nanjing Jiancheng Co., Ltd., Nanjing, China) according to the manufacturer's instructions. The colon tissue samples were prepared as follows: colon tissue was accurately weighed, and then physiological saline was added as the homogenization medium in a weight-to-volume ratio of 1:19. A 5% tissue homogenate was then prepared in a tissue-dispersing machine for MPO activity determination. MPO activity was measured in U/g of fresh colon tissue, with one unit of MPO defined as the amount required to degrade 1.0 mmol of hydrogen peroxide per minute at 37 C.

H&E staining
The colon morphology and histopathological lesions were assessed with H&E staining (Murthy, Cooper, & Shim, 1993). The colon tissues were first cut into 5-mm-thick slices. The slices were then fixed in neutral buffered formalin for 24 h, dehydrated with graded alcohol (75%-100%) solutions, and then embedded in paraffin wax for H&E staining. The severity of colonic histological injury in each mouse was scored using a modified scoring system that took into account the degree of inflammation, mucosal damage, crypt damage, and range of pathological changes.

RNA extraction and RT-qPCR
Total RNA was extracted from each colon tissue sample using TRIzol reagent (Shenggong bio, Shanghai, China). cDNA was then prepared by reverse transcription using a HiScript III RT SuperMix for qPCR (Vazyme Biotech, China) and amplified by RT-qPCR using Hieff qPCR SYBR Green Master Mix (Yeasen Biotech, China) and the appropriate primers ( Table 1). The conditions were 40 cycles of 95 C for 30 s, 95 C for 5 s, and 60 C for 30 s. The primers used in this study were synthesized by Beijing Genomics (Shanghai, China). The RNA expression levels of the relevant genes in each group were measured using the 2 -DDCt method, where D Ct represents the difference in the Ct values between the target gene and the b-actin reference gene. The b-actin mRNA levels in the test groups are expressed as the ratio of its expression in the test group relative to that in the DSS group.

Fecal TBAs concentration analysis
Faecal samples were prepared for TBA analysis. Mice faeces that were stored at -80 C were thawed in an ice bath to reduce sample degradation, 100 mg/mL of feces in each group were homogenate with ethanol, which were centrifuged at 4 C for 10minat 12000 rpm, and the supernatant were collected and added to the reagent according to the instructions of TBA determination kit (TONGWEI, CHINA), the optical density at 405 nm was measured after 3 min for reaction, and content of TBA was calculated by the following equation.