The effect of synbiotics Bifidobacterium infantis and milk oligosaccharides on shaping gut microbiota community structure and NASH treatment

Probiotic Bifidobacterium longum subspecies infantis (Bifidobacterium infantis) consumes human milk oligosaccharides (MO) and protects intestinal permeability thereby having anti-inflammatory effects (Underwood et al., 2015; Bode, 2006; Asakuma et al., 2011) [1–3]. Via the gut-liver axis, gut barrier disruption and dysbiosis lead to hepatic inflammation (Sheng et al., 2017; Jena et al., 2017) [4,5,6]. Our published data revealed that butyrate, as well as synbiotics of B. infantis in combination with MO, had protective effects against cancer-prone non-alcoholic steatohepatitis (NASH) mouse models, i.e., Western diet (WD)-fed bile acid receptor FXR (farnesoid x receptor) knockout (KO) mice (Jena et al., 2018) [6,7]. In addition, MO was particularly effective in increasing the blooming of butyrate-generating bacteria (Jena et al., 2018) [7]. In the present study, we further showed that the reduced ileal short chain fatty acid (SCFA) signaling found in WD-fed FXR KO mice could be reversed by B. infantis and/or MO treatment. Moreover, ileal mRNA levels of SCFA receptors i.e. Gpr41 (Ffar3), Gpr109 (Hcar2), and Gpr43 (Ffar2) were increased in B. infantis and/or MO-treated mice suggesting increased SCFA signaling (Fig. 1). Further, nuclear magnetic resonance (NMR) data revealed that MO and B. Infantis plus MO increased intestinal acetate, propionate, butyrate, and valerate levels (Fig. 2). In addition, B. infantis and/or MO reduced the abundance of genus Bilophila and the relative copy number of bacterial genes including dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase A (mcrA), which were all increased in cancer-prone FXR KO mice (Fig. 3).

suggesting increased SCFA signaling (Fig. 1). Further, nuclear magnetic resonance (NMR) data revealed that MO and B. Infantis plus MO increased intestinal acetate, propionate, butyrate, and valerate levels (Fig. 2). In addition, B. infantis and/or MO reduced the abundance of genus Bilophila and the relative copy number of bacterial genes including dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase A (mcrA), which were all increased in cancer-prone FXR KO mice (Fig. 3).
& 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology, Microbiology, Molecular Biology More specific subject area Synbiotics Type of data Value of the data -In our earlier report, B. infantis and MO treatment reduced hepatic and ileal inflammation [7]. It has been shown that B. infantis and MO protects from gut barrier function and inflammation [1][2][3]. In addition, the abundance of bacterial butyrate-generating genes bcoA (butyryl-CoA: acetate CoAtransferase) and buk (butyrate kinase) in the cecum was increased with MO and B. infantis plus MO treatment. Data in this study described the mRNA expression of SCFA receptors regulated by B. infantis and MO treatment. Our data showed that increased SCFA signaling with B. infantis and MO treatment was associated with reduced inflammation. -In our earlier report, the concentration of butyrate was reduced in FXR KO mice [6]. Data in this study described that B. infantis and MO increased SCFAs, i.e., acetate, propionate, butyrate, and valerate. Additionally, such increases were accompanied by reduced inflammation in FXR KO mice. It has been shown that dysregulated bile acid and gut dysbiosis causes hepatic inflammation [4][5][6][7]. Moreover, the abundance of genus Bilophila and dsrA gene, which are involved in the generation of hydrogen sulfide, was increased by FXR inactivation and B. infantis and MO treatment reversed those changes. -These data suggested that the anti-inflammatory effect of B. infantis and MO may due to increased SCFAs and reduced hydrogen sulfide and methane.

Data
The data showed the mRNA level of ileal SCFA receptors i.e. Gpr41, Gpr109a, and Gpr43 in WD-fed wild type (WT) mice and WD-fed FXR KO mice supplemented with or without Bifidobacterium infantis and/or MO (Fig. 1). NMR data generated from cecal content showed the concentration of acetate, propionate, butyrate, and valerate (Fig. 2). Moreover, bacteria and their functional genes were determined by real-time quantitative PCR (Fig. 3).

Mice
Specific pathogen-free male C57BL/6 WT mice (Jackson Laboratory, Sacramento, CA, USA) and FXR KO mice were housed in steel microisolator cages at 22°C with a 12-h light/dark cycle. They were given a WD containing 21.2% fat, 34% sucrose, and 0.2% cholesterol (Envigo, Indianapolis, IN, USA) right after weaning (3 weeks, 6-10 mice per group). When mice were 3-month old, they were given B. infantis (10 9 cfu per mouse, orally, once a week) in saline, bovine MO (7% in a diet that lacks 7%

Gene expression and quantification of bacterial genes
RNA was isolated from mouse tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA. qRT-PCR was performed on an ABI 7900HT Fast real-time PCR system using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The mRNA levels were normalized to the level of Gapdh mRNA. The primer sets used are described in Table 1.
For bacterial gene quantification, cecal (0.05 g) DNA was extracted using ZR Fecal DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA), quantified by NanoDrop (Thermo Scientific, Wilmington, DE, USA), and amplified using primers listed in Table 1. A dissociation step was included to analyze the melting profile of amplified products. In parallel, qPCR was performed using ten-fold serial diluted synthetic DNA fragments (Integrative DNA technologies, Redwood city, CA, USA) of a bacterial gene with known concentrations. Bacterial DNA concentration was calculated using standard curves of diluted synthetic DNA fragment based on method described elsewhere [8].

1 H NMR spectroscopy for metabolic profiling
Cecal samples (50 À 60 mg) were prepared and analyzed by 1 H NMR based on published method [6,9]. Each cecal sample was homogenized and mixed with sodium-potassium phosphate buffer and centrifuged at 14,000g for 20 min. All 1 H NMR spectra were collected using a Bruker advance 600  Table 1 Primers used for qPCR.

Gene
Forward qPCR primers used to quantify gene expression in mice GGCTTCTACAGCAGCATCTA AAGCACACCAGGAAATTAAG qPCR primers used to quantify bacterial genes Bilophila (tpA) CGGTATCGAAATCGTGAAGG CAGAGGGTCAGGGTGTTGTT dsrA GCCGTTACTGTGACCAGCC GGTGGAGCCGTGCATGTT mcrA TTCGGTGGATCDCARAGRGC GBARGTCGWAWCCGTAGAATCC NMR spectrometer (Bruker; Rheinstetten, Germany). The samples were pre-cooled to 277 K before being loaded into the magnet, and were warmed to 298 K and equilibrated for 5 min before data acquisition. For each sample, 64 scans were collected into 64 K data points over a spectral width of 20 ppm with a relaxation delay of 2 s. Identification and quantification of metabolites were accomplished using Chenomx NMR Suite 7.6 (Chenomx, Inc., Edmonton, Alberta, Canada).

Statistical analysis
Data are presented as the mean 7 SD. Statistical analysis was performed by unpaired Student's t-test or one-way analysis of variance by using GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA). P o 0.05 was considered statistically significant.