High-fat diet-mediated dysbiosis exacerbates NSAID-induced small intestinal damage through the induction of interleukin-17A

Non-steroidal anti-inflammatory drugs (NSAIDs) cause damage in the small intestine in a bacteria-dependent manner. As high-fat diet (HFD) is a potent inducer of gut dysbiosis, we investigated the effects of HFD on bacterial flora in the small intestine and NSAID-induced enteropathy. 16S rRNA gene analysis revealed that the population of Bifidobacterium spp. significantly decreased by fold change of individual operational taxonomic units in the small intestine of mice fed HFD for 8 weeks. HFD increased intestinal permeability, as indicated by fluorescein isothiocyanate-dextran absorption and serum lipopolysaccharide levels, accompanied by a decrease in the protein expressions of ZO-1 and occludin and elevated mRNA expression of interleukin (IL)-17A in the small intestine. HFD-fed mice exhibited increased susceptibility to indomethacin-induced damage in the small intestine; this phenotype was observed in normal diet-fed mice that received small intestinal microbiota from HFD-fed mice. Administration of neutralizing antibodies against IL-17A to HFD-fed mice reduced intestinal permeability and prevented exacerbation of indomethacin-induced damage. Thus, HFD-induced microbial dysbiosis in small intestine caused microinflammation through the induction of IL-17A and increase in intestinal permeability, resulting in the aggravation of NSAID-induced small intestinal damage.

increase in intestinal permeability in HfD-fed mice. Small intestinal permeability was examined by measuring fluorescein isothiocyanate (FITC)-dextran and serum lipopolysaccharide (LPS) that is a membrane component found in gram-negative bacteria. Serum FITC-dextran in the HFD group was significantly elevated by 2.0-fold prior to indomethacin administration compared with that in the control group (p = 3.3 × 10 −4 ; Fig. 3A). This result was consistent with changes in serum LPS levels (p = 5.2 × 10 −3 ; Fig. 3B).
Effects of HFD treatment on the expressions of inflammatory cytokines and tight junction components. We measured the expressions of inflammatory cytokines, innate immune receptors, and tight junction components in the small intestine of HFD-fed mice prior to indomethacin administration. The mRNA levels of interleukin (IL)-17A (Fig. 4A) and MCP-1 (Fig. 4B) in the HFD group were significantly increased by 3.5-fold (p = 7.8 × 10 −3 ) and 1.5-fold (p = 3.3 × 10 −3 ), respectively, although there were no differences in the expressions of other inflammatory cytokines, such as IL-1β, TNF-α, and toll-like receptor (TLR) 4 ( Fig. 4C-E). The mRNA levels of TLR 9 decreased significantly in the HFD group (p = 3.8 × 10 −2 ; Fig. 4F), and the expressions of tight junction proteins, such as ZO-1 (Fig. 4H) and occludin (Fig. 4I), were reduced in the small intestinal mucosa in the HFD group.

Effects of neutralizing IL-17A on indomethacin-induced small intestinal damage in HFD-fed mice.
To determine the role of IL-17A, we administered anti-IL-17A antibody or isotype control to HFD-fed mice prior to indomethacin treatment once daily for 7 days. Two days after the final dose, we administered indomethacin (12 mg/kg BW) by gavage, and the mice were sacrificed 24 h later. Lesion index was significantly reduced in the HFD group treated with anti-IL-17A antibodies (p = 9.3 × 10 −3 ; Fig. 5A), and this result was consistent with the histological observations (p = 8.0 × 10 −4 ; Fig. 5B). Furthermore, IL-17A neutralization significantly reduced intestinal permeability and serum LPS in the HFD group (p = 1.4 × 10 −2 ; Fig. 5C; p = 3.5 × 10 −2 ; Fig. 5D).
transplantation of altered small intestinal microbiota from HfD-fed mice. To investigate whether microbiota modulates indomethacin-induced small intestinal damage, small intestinal luminal contents collected from donor mice fed HFD-60 or AIN-93M were transplanted into microbiota-depleted recipient mice, treated with antibiotic cocktail, fed AIN-93M once daily for 5 days. One day after the final transplantation, mice were administered indomethacin (12 mg/kg BW) by gavage and sacrificed 24 h later (Fig. 6A). As a result, intestinal microbiota transplantation from HFD-60-fed mice exacerbated small intestinal damage, and significantly increased lesion index (p = 1.1 × 10 −2 ; Fig. 6B) and histological score (p = 1.1 × 10 −3 ; Fig. 6C) were observed.

Discussion
In this study, we demonstrated that HFD treatment changed the gut microbiota in the small intestine and exacerbated NSAID-induced small intestinal damage. As the small intestinal microbiota plays an underlying role in the regulation of endotoxemia and inflammatory response, it is essential to identify major bacteria present in the small intestine of HFD-treated mice. Microbiota analysis showed that the proportion of gram-positive anaerobic bacilli, Bifidobacterium spp., was significantly reduced in HFD-fed mice. It has been reported that PPI-treatment exacerbated NSAID-induced small intestinal injury by inducing the reduction of Bifidobacteria spp., and recolonization with Bifidobacteria-enriched commensal bacteria prevented the damage 4 . Bifidobacterium spp. do not degrade intestinal mucus glycoproteins, but, instead, promote a stable environment and improve gut barrier function by inhibiting the translocation of pathogenic enterobacteria and endotoxin 11,12 . By contrast, increasing the proportion of gram-negative bacteria can decrease the integrity of intestinal mucosa and lead to metabolic endotoxemia 13 . In this study, it is assumed that a decrease in Bifidobacterium spp. was involved in intestinal hyperpermeability and metabolic endotoxemia observed in HFD-fed mice.
Intestinal permeability is regulated by the components of the epithelial cellular tight junction, such as ZO-1 and occludin. In this study, the protein expressions of ZO-1 and occludin decreased in the small intestinal mucosa of HFD-fed mice. The dysfunction of intestinal tight junction barrier is associated with intestinal microbiota dysbiosis 14 . Hence, we proposed that the disruption of intestinal tight junction components in this study was accelerated by HFD-induced dysbiosis, which exacerbated intestinal permeability. Consequently, endotoxin was increased in the blood, triggering a cascade of inflammatory responses. In a previous study, the administration of NSAID accelerated intestinal permeability during absorption 15 and resulted in the induction of mucosal damage. In the present study, we assume that HFD-induced intestinal barrier dysfunctions facilitated enterobacterial invasion of mucosae and metabolic endotoxemia, which may have promoted the development of indomethacin-induced small intestinal damage.
We previously reported that when mucosal barrier function is disrupted, LPS and high-mobility group box 1 from the injured epithelial cells bind to TLR 4 and activate NF-κB, through the MyD88-dependent pathway www.nature.com/scientificreports www.nature.com/scientificreports/ and the NLR family pyrin domain-containing 3 inflammasome. This leads to the release of proinflammatory cytokines, such as TNF-α and IL-1β, in NSAID-induced small intestinal damage [16][17][18] . Although the mRNA levels of TLR4, TNF-α, and IL-1β did not change, the mRNA level of TLR9 decreased in small intestine treated with HFD before NSAID administration. TLR9 is involved in the activities of macrophages against bacterial infection 19 by identifying unmethylated CpG-DNA sites in bacterial DNA 20 . It is considered a protective factor in intestinal homeostasis, and Western diets are associated with its dysfunction 21 . Long-term administration of HFD in this study may have reduced the expression of TLR9 resulting in the deactivation of its protective function in this study.
We demonstrated that IL-17A was elevated in HFD-treated small intestine, and the neutralization of IL-17A improved the indomethacin-induced damage, intestinal permeability, and characteristics of endotoxemia. IL-17A is a pro-inflammatory cytokine associated with a variety of inflammatory diseases 22 , and there are several reports indicating that HFD intake induces the expression of IL-17 and exacerbates inflammatory diseases, e.g., psoriasis, colitis, and steatohepatitis [23][24][25] . Recent studies suggested that HFD-induced microbial alterations, especially the expansion of Firmicutes 26,27 , might be responsible for IL-17 induction. It is possible that the increased populations of Clostridiales and Erysipelotrichaceae belonging to Firmicutes in the HFD group might have enhanced the expression of IL-17A, thereby promoting small intestinal microinflammation and intestinal permeability in this study. Our result aligns with that of a previous study, which demonstrated that gut epithelial barrier dysfunctions can be reversed by IL-17A neutralization in a neuro-psychoactive drug-induced bacterial infection model 28 . Therefore, a therapeutic approach targeting IL-17A may help to diminish the metabolic burden of endotoxemia caused by defective intestinal barrier functions. A deeper mechanistic understanding of how IL-17A may interact with microbiota for homeostatic control of intestinal epithelia is warranted for the development of a novel therapeutic strategy against NSAID-induced small intestinal damage.
In previous studies, it has been demonstrated that intestinal microbiota is involved in the pathogenesis of NSAID-induced enteropathy [29][30][31] . Therefore, drugs regulating intestinal flora, such as probiotics, can be utilized as therapeutic agents 32 . It is known that Clostridium difficile-induced colitis can be remarkably restored by changing intestinal flora through feces transplantation from healthy subjects into the large intestine of patients; hence, fecal microbiota transplantation (FMT) can be applied to various other intestinal diseases 33 . There is a scarcity of FMT studies on the flora of small intestine as most FMT studies have examined the bacterial flora of large intestine. In this study, small intestinal microbial transplantation from HFD-fed mice exacerbated indomethacin-induced small intestinal damage. The alterations of small intestinal microbiota with HFD treatment may have increased the susceptibility of mucosae and they were sufficient to influence on indomethacin-induced enteropathy. To the best of our knowledge, this is the first report demonstrating that HFD-induced dysbiosis forms the substrate to aggravate indomethacin-induced enteropathy through IL-17A.
In conclusion, HFD-induced alterations of small intestinal microbiota cause microinflammation through the induction of IL-17A, and an increase in intestinal permeability, which aggravate NSAID-induced small intestinal damage. Low-fat dietary therapy and strategies to enhance individual-specific innate functions of Bifidobacterium may be useful for the prevention and mitigation of NSAID-induced small intestinal damage. A therapeutic strategy targeting IL-17A could be a treatment option for such enteropathy.

Methods
Animals and diets. Specific pathogen-free male C57BL/6 J mice (4 weeks old, 10-15 g) were purchased from Charles River Japan Inc. (Atsugi, Japan). Animals were housed in polycarbonate cages with paper-chip bedding in an air-conditioned biohazard room with a 12-h light/dark cycle. All animals had free access to food and water. Mice were acclimated for 1 week prior to the experiments and received regular chow (CE-2, CLEA Japan Inc., Tokyo, Japan). At 6 weeks of age, mice were randomly separated into a control group fed AIN-93M (Oriental www.nature.com/scientificreports www.nature.com/scientificreports/ Yeast Co., Tokyo, Japan) or an HFD group fed HFD-60 (Oriental Yeast Co.) for 8 weeks. The composition of AIN-93M and HFD-60 is shown in Supplementary Table S1.
All experiments were carried out under the control of animal research committee in accordance with the Guidelines on Animal Experiments in Osaka City University Graduate School of Medicine, the Japanese Serial 5-μm-thick cryostat sections were mounted on silanized slides (Dako, Tokyo, Japan) and stained with hematoxylin and eosin. For each mouse, at least ten random villi at the damaged areas were scored in a masked fashion by 2 investigators independently. For quantitative evaluation, a modified histological scoring system was used 18 . Total histology scores ranged from 0 to 13 and were evaluated for 6 categories with sub-scores as follows: epithelium (0, normal; 1, flattened; 2, loss of epithelial continuity; 3, severe denudation), villus shape (0, normal; 1, short and rounded; 2, extremely short and thick), villus tip (0, normal; 1, damaged; 2, severely damaged), stroma (0, normal; 1, slightly retracted; 2, severely retracted), inflammation (0, no infiltration; 1, mild infiltration; 2, severe infiltration), and crypt status (0, normal; 1, mild crypt loss; 2, severe crypt loss). www.nature.com/scientificreports www.nature.com/scientificreports/ DNA extraction and 16S rRNA gene analysis. Small intestinal luminal contents were collected immediately after sacrifice, using a feces collection kit (Takara Bio, Shiga, Japan). Genomic DNA was then isolated using the NucleoSpin Microbial DNA Kit (MACHEREY-NAGEL, Düren, Germany). Approximately 500 µl of stored small intestinal samples were placed in a microcentrifuge tube containing 100 µl elution buffer. The mixture was placed into the NucleoSpin Beads Tube with proteinase K, which was subjected to mechanical beads-beating for 12 min at 30 Hz in the TissueLyzer LT. DNA extraction was subsequently performed as per manufacturer's instructions. DNA samples were purified using the Agencourt AMPure XP (Beckman Coulter, Brea, CA).
V3-4 amplicon sequences of 16S rRNA genes were prepared as described previously 34 . Briefly, 16S rRNA gene fragments including V3 and V4 regions were amplified by PCR (forward primer: ACACGAC GCTCTTCCGATCTCCTACGGGNGGCWGCAG, reverse primer: GACGTGTGCTCTTCCGATC TGACTACHVGGGTATCTAATCC) for 20 cycles, and PCR products were purified with Agencourt AMpure beads (Beckman Coulter). Next, index sequences for sequencing were added by running a second PCR with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set1, New England Biolabs) for 8 cycles, and the products were purified with Agencourt AMpure beads. For each sample, an equal amount of each DNA amplicon library was mixed and sequenced on the MiSeq instrument (Illumina) using a MiSeq v3 Reagent kit with 20% PhiX (Illumina). www.nature.com/scientificreports www.nature.com/scientificreports/ fitc-Dextran permeability assay. Intestinal permeability was measured as previously described with some modifications 10 . Mice were fasted for 16 h and FITC-dextran was administered by gavage (500 mg/kg BW, 125 mg/ml; Sigma-Aldrich). After 4 h of administration, 120 μl of blood was collected from the heart, stored on ice in the dark, and centrifuged (15,000 rpm) at 4 °C for 10 min. Serum was diluted in an equal volume of PBS (pH 7.4) and analyzed for FITC-dextran concentration with a fluorescence spectrophotometer (Varioskan LUX; Thermo Scientific, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
Blood sampling and LPS assay. Blood samples were obtained by cardiac puncture and stored on ice for 30 min before centrifugation (15,000 rpm) at 4 °C for 10 min. Serum portions were isolated, and LPS levels were measured with the murine LPS ELISA kit (Cusabio, Wuhan, China).
Real-time quantitative RT-PCR. Total RNA was extracted from small intestinal tissues with the ISOGEN kit (Nippon Gene Co., Ltd., Tokyo, Japan). Real-time quantitative RT-PCR analyses were performed as previously described 35 . The sequences of PCR primers and TaqMan probes of ZO-1, MCP-1, IL-1β, TNF-α, and TLR 4 are shown in Supplementary Table S2. TaqMan Gene Expression Assays (Life Technologies Corp., Rockville, MD, USA) were used for measuring occludin, IL-17A, and TLR9. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and mRNA levels were expressed as ratios relative to the mean of control mouse data.
Small intestinal microbiota transplantation. After 8 weeks on control diet, recipient mice were given drinking water containing ampicillin (1 g/l), metronidazole (1 g/l), neomycin (1 g/l), and vancomycin (0.5 g/l) for 2 weeks. Small intestinal luminal contents from donor mice were collected, dissolved in 10 g/L sterile PBS, centrifuged at 1,500 rpm for 5 min. Supernatants were administered to the recipient mice by gavage (0.5 ml/mouse) for 5 days. After the final day of transplantation, mice were administered indomethacin (12 mg/kg BW; Sigma-Aldrich) in a 0.5% carboxymethylcellulose solution by gavage and sacrificed 24 h later.

Statistical analysis.
Values are expressed as mean ± standard error of the mean (SEM) in animal experiments. Comparisons between 2 groups were performed using a two-sided Student's t-test. ANOVA was used to compare differences among multiple groups, and post-hoc analysis was performed by Tukey's multiple comparisons test. P-values < 0.05 indicate a statistical significance.