F. prausnitzii ATCC27768 (purchased from ATCC) was grown under anaerobic conditions with N₂ gas using the Hungate method (35). The culture medium contained the following contents per liter: 10 g Bacto peptone, 10 g LAB-LEMCO powder, 3 g Bacto yeast extract, 5 g D-glucose, 2.5 g maltose, 5 g NaCl, 1 g soluble starch, 1 g L-cysteine HCl, 3 g sodium acetate and 1 mg resazurin. F. prausnitzii was cultured at 37°C until the optical density at 650 nm reached >1.5. Prior to administration to mice, bacterial culture fluid was collected anaerobically with a 1 ml syringe, and was stored at 4°C until administration. Inactivation of the bacterium was performed by air mixing. Culture fluid (30 ml) was transferred from the Hungate tube to a 50-ml centrifugation tube, gently mixed well without using a vortex mixer and exposed to air for at least 30 min. Following air mixing, one tube was used as inactivated F. prausnitzii (iFP), and another was divided into bacterial cells (FP cells) and supernatant (FP sup.).

All the experimental procedures using mice were conducted in accordance with the National Institutes of Health guidelines for the use of experimental animals (36). Experiments were performed according to the guidelines for the care and use of laboratory animals approved by Nichinichi Pharmaceutical Co., Ltd. All experimental programs were approved by the Animal Care Committee of Kyoto Prefectural University of Medicine (Kyoto, Japan). BALB/cCrSlc mice (5-week-old males) were purchased from Japan SLC, Inc. A total of 100 mice were used in this study. They were acclimatized for 1 week prior to experiments, and were housed individually in a room maintained at 22°C and normal humidity, under a 12:12-h day/night cycle throughout the experiments. The mice were allowed free access to rodent chow (CE-2; CLEA Japan, Inc.) and filtered tap water. Experimental colitis was initiated by oral administration of 5% DSS (molecular weight, 5,000 Da) mixed with tap water. Body weight was measured daily throughout every experiment. The mean body weight was 21.1±0.8 g following acclimatization. Health and behavior were monitored daily by measuring body weight. Mice were euthanized within 1 h if the body weight decreased to <80% of the weight at the start of experiments. No animals succumbed prior to meeting the criteria for sacrifice. No other measures were implemented due to the minor distress that may be induced. Research staff were trained in animal care and handling by an animal experiment specialist.

A total of two preventive experiments were performed. In the first experiment, mice were divided into 4 groups: Control mice (Cont. group, n=9); F. prausnitzii-treated mice (FP group, n=8); 5% DSS-treated mice (DSS + Cont. group, n=19); and 5% DSS plus F. prausnitzii-treated mice (DSS + FP group, n=20). To determine the experimental preventive effects of F. prausnitzii on colitis, 0.2 ml/mouse/day F. prausnitzii culture fluid containing 10⁸ colony forming units (CFU) of live F. prausnitzii was administered orally for 7 days, and then DSS was administered for 8 days. F. prausnitzii was not administered during the DSS treatment period. The mice were sacrificed at day 8 by cervical dislocation to minimize suffering. The large intestine of each mouse was removed, and the length from under the cecum to the anus was measured as the colon length. H&E was used to stain colonic mucosa from mice in the DSS + Cont. (n=2) and DSS + FP groups (n=2). Staining was 0.2% hematoxylin solution for 5 min, rinsing in tap water then 1% acid alcohol, rinsing in tap water again, rinsing in ethanol, staining for 5 min with 1% eosin solution, and finally rinsing in ethanol. This was all performed at room temperature Reverse transcription-quantitative PCR (RT-qPCR) analysis was performed on 5 samples/group.

In the second experiment, the effects of inactivated culture fluid, bacterial cells and supernatant were determined using the same methods. Mice were divided into 5 groups: Control mice (Cont. group, n=5); 5% DSS-treated mice (DSS + cont. group, n=4); 5% DSS plus inactivated F. prausnitzii-treated mice (DSS + iFP group, n=4); 5% DSS plus F. prausnitzii cell-treated mice (DSS + FP cell group, n=5); and 5% DSS plus F. prausnitzii supernatant (DSS + FP sup. group, n=5). Group details are presented in Table I. In the preventive experiment, as one animal in the DSS + Cont. group reached the endpoint criteria, it was sacrificed and its data were excluded; the weight loss was hypothesized to be a result of weakness due to DSS-induced colitis.

To investigate the therapeutic effects of F. prausnitzii on colitis, mice were administered 5% DSS water for 8 days following the acclimation period to induce DSS-colitis. Administration of 5% DSS water was then terminated for a recovery period of 3 days. Then, 0.2 ml/mouse/day of F. prausnitzii culture fluid for the DSS + FP group (n=10), or fresh culture medium for the DSS + Cont. group (n=10) was administered orally for 7 days. The mice were sacrificed on day 19 by cervical dislocation to minimize suffering. Histological analyses using H&E staining as above was performed on 5 samples/group. RT-qPCR analysis was performed on 5 samples/group.

Mucosal inflammation was assessed using the disease activity index (DAI) score, as illustrated in Table II. The DAI scores were calculated by the sum of the fecal properties and hematochezia scores. The DAI score was measured daily.

For histological analysis, the colon was fixed in 10% buffered formalin at room temperature for ~1 week and embedded in paraffin. Sections (4-µm thickness) were prepared and stained with hematoxylin and eosin as above. The severity of histological damage was scored in a blinded manner using a previously published grading system (37,38). Briefly, the histological scores indicated the presence and extent of inflammation and epithelial damage in colitis, as determined according to the system presented in Table III. The total score indicated the sum of the epithelium (E) and infiltration (I) scores, and thus ranged between 0 and 8 (total score=E+I).

Total RNA was extracted from the mucosa of the large intestine using the acid guanidinium phenol chloroform method with Isogen (Nippon Gene Co., Ltd.) and then reverse transcribed with PrimeScript™ RT-PCR kit (Takara Bio, Inc.). The reverse transcription conditions were: 65°C for 5 min (annealing step), 42°C for 30 min (reverse transcription step), and then 95°C for 5 min (inactivation step). cDNA was subjected to qPCR on the 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.). qPCR conditions were: 95°C for 15 sec, then 40 cycles of 95°C for 15 sec and 60°C for 31 sec. The expression of cytokine mRNA [tumor necrosis factor-α (TNFα); interferon-γ (IFNγ); interleukin (IL)-10, IL-12 and IL-17A] was determined; β-actin was used as an internal control. The 2^−∆∆Cq method was used for quantification (39). The sequences of the primer sets are presented in Table IV.

Data were analyzed using StadView version 5.0 (SAS Institute, Inc.). DAI scores over time and mice body weight changes over time were compared by one-way ANOVA followed by Fisher's protected least significant difference test. Student's t-test was used for comparing two groups. P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard deviation for all experiments with the exception of RT-qPCR data, which are presented as the mean ± standard error of the mean.

The administered F. prausnitzii fluid (0.2 ml) contained ~1.5×10⁸ CFU. All mice administered DSS developed diarrhea, bloody stools, weight loss and shortening of the colon, all symptoms of colitis (Figs. 1–4); however, these symptoms were alleviated in mice administered living F. prausnitzii prior to DSS administration (DSS + FP groups). Diarrhea and bloody stools were assessed using the DAI score; this value was significantly improved in the DSS + FP group compared with the DSS + Cont. group from day 6 onwards (P<0.01; Fig. 1). Weight loss was also significantly alleviated in the DSS + FP group compared with the control (Fig. 2). DSS + FP group mice exhibited similar weight loss to the DSS + Cont. group until day 6; however, from day 7, the relative weights of the mice were significantly increased in the DSS + FP group compared with the DSS + Cont. group. The weight of the DSS + Cont. group by day 8 was reduced to 86.2±4.2% of the starting weight, whereas that of the DSS + FP group was reduced to 89.7±5.2%. The colon length of the DSS+FP group was significantly increased compared with the DSS + Cont. group (Fig. 3). The colon length of the DSS + Cont. group was reduced by 20.8±8.8%, whereas that of the DSS + FP group was decreased by 15.2±6.8%. It was observed that the administration of inactivated F. prausnitzii induced no protective effects against DSS-induced colitis in BALB/c mice. The DAI score, weight loss and colon length reduction were not ameliorated in mice administered inactivated bacterial materials (Fig. 4). In the absence of DSS treatment, when comparing the FP group and the Cont. group, no notable side effects of F. prausnitzii administration on weight change, DAI score, colon length, appearance or behavior were observed.

The internal folds in the colons of mice administered DSS were reduced by inflammation (Fig. 5). The histological scores of the DSS + FP groups were significantly improved compared with the DSS + Cont. groups (P<0.05). Inflammation of the colon was alleviated in mice administered DSS with preventive F. prausnitzii compared with mice administered DSS alone.

The expression of TNFα, IFNγ, IL-17A, IL-10 and IL-12 normalized to β-actin in the mucosa of the large intestine is presented in Fig. 6. There were no significant differences in the expression of each cytokine upon comparing the DSS + FP group with the DSS + Cont. group; however, the expression levels of TNFα, IFNγ and IL-10 in the DSS + FP group were notably decreased compared with the DSS + Cont. group. The levels of IL-17A and IL-12 were markedly unaltered in the DSS + FP group compared with the DSS + Cont. group.

For the therapeutic experiment, animals were first administered DSS, and then control or FP treatment. There were no significant differences between the DSS + Cont. and DSS + FP groups in terms of DAI score, weight change and colon length; however, the histological score of animals in the DSS + FP group was significantly improved (Fig. 7). Proinflammatory cytokine expression was measured by RT-qPCR. The expression of all cytokines analyzed during the experiment was markedly downregulated in the mucosa of the DSS + FP group compared with the DSS + Cont. group; however there were no significant differences (Fig. 8).