5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium

5-ASA checks the growth of E. coli during DSS-induced colitis.

We first determined whether the ability of 5-ASA to reduce the abundance of E. coli in the fecal microbiota of ulcerative colitis patients (23) could be recapitulated during DSS-induced colitis in mice. To establish an animal model for addressing this question, we used C57BL/6J mice from The Jackson Laboratory, which do not carry endogenous Enterobacterales (26), because the vendor screens against the presence of this taxon in its special-pathogen-free procedures. The fact that the ecological niche of Enterobacterales remains unoccupied during microbiota assembly in C57BL/6J mice offers the unique opportunity for precision editing of the microbiota by the designed engraftment of E. coli strains engineered to probe the contribution of predestined metabolic pathways to bacterial growth. Since previous studies suggested that an E. coli expansion in DSS-treated mice required genes for the respiration of host-derived oxygen and nitrate (25, 27, 28), C57BL/6J mice that were confirmed to be Enterobacterales free were engrafted with a 1:1 mixture of a respiration-proficient E. coli strain (Nissle 1917, wild type) and an isogenic mutant lacking genes for aerobic and nitrate respiration (cydA napA narG narZ mutant). Differential recovery of these strains serves as an indicator for the bioavailability of respiratory electron acceptors in the colon (29). DSS treatment of male and female mice was associated with reduced colon length (see Fig. S1A in the supplemental material) and a marked E. coli expansion (Fig. S1B), which required genes for aerobic and nitrate respiration, as indicated by increased recovery of the wild type over that of the cydA napA narG narZ mutant from DSS-treated mice in a comparison with controls (Fig. S1C).

Having established a model to study an expansion of E. coli during DSS-induced colitis, we wanted to determine whether the growth of this species could be limited by 5-ASA treatment. DSS treatment resulted in marked colitis, as indicated by a reduction in colon length (Fig. 1A) and marked histopathological lesions (Fig. 1B), including crypt hyperplasia, an epithelial repair response, and lower numbers of alcian-blue positive (goblet) cells (Fig. S2A and S2B), which is reflective of a reduction in the number of terminally differentiated cells during epithelial repair. However, these signs of disease were blunted in animals receiving 5-ASA supplementation (Fig. 1A and B; Fig. S2A and S2B). DSS treatment resulted in a marked expansion of E. coli in the colonic microbiota, which was blunted by 5-ASA treatment (Fig. 1C). As in a previous report (24), 5-ASA reduced the growth of E. coli under aerobic conditions in vitro (Fig. S2C). However, 5-ASA did not inhibit the growth of E. coli under anaerobic conditions (Fig. S2D), suggesting that environmental factors markedly influenced the inhibitory activity of 5-ASA, thus making in vitro growth assays a poor predictor of whether the drug would inhibit bacterial growth in the intestine in vivo. Notably, 5-ASA supplementation in mock-treated mice did not reduce the overall recovery of E. coli from colon contents compared to that from mock-treated mice without 5-ASA supplementation (Fig. 1C), which did not support the idea that 5-ASA directly acts on bacteria to block the growth of E. coli in the colon (24). In conclusion, 5-ASA treatment checked an E. coli expansion in mice with DSS-induced colitis, thus recapitulating a reduction in the E. coli abundance observed in ulcerative colitis patients during 5-ASA therapy (23).

Elevated recovery of the respiration-proficient E. coli wild type over the respiration-deficient cydA napA narG narZ mutant was consistent with previous reports indicating that DSS-induced colitis increases the luminal bioavailability of oxygen and nitrate (25, 27, 28), but this increase in the competitive index was blunted in DSS-treated mice receiving 5-ASA supplementation (Fig. 1D). These data suggested that 5-ASA supplementation checks an E. coli expansion by limiting the availability of host-derived electron acceptors. The colonic epithelium is a potential source of host-derived oxygen, because epithelial oxygenation increases during DSS-induced colitis (28). Visualization of epithelial oxygenation with pimonidazole, a 2-nitroimidazole that is reductively activated specifically in hypoxic cells (<1% oxygen) (30, 31), revealed that DSS treatment eliminated epithelial hypoxia in the colon, whereas 5-ASA supplementation restored epithelial hypoxia in DSS-treated mice (Fig. 1E and F). Since PPAR-γ maintains epithelial hypoxia in the colon by increasing oxygen consumption in the mitochondria (29), we investigated whether 5-ASA supplementation restored markers of mitochondrial bioenergetics in the colonic epithelium. Consistent with impaired mitochondrial bioenergetics, DSS treatment reduced epithelial ATP levels (Fig. 1G) and decreased epithelial levels of pyruvate dehydrogenase activity (Fig. 1H). Levels of these markers of mitochondrial bioenergetics could be restored in the colonic epithelium by 5-ASA treatment (Fig. 1G and H). To directly address the contribution of oxygen, mice were engrafted with a 1:1 mixture of a respiration-proficient E. coli strain (Nissle 1917, wild type) and an isogenic mutant lacking cydA, the gene encoding cytochrome bd oxidase, which is required for aerobic respiration under microaerophilic conditions (29). DSS-induced colitis (Fig. S2E) provided a cydA-dependent fitness advantage, which was blunted by treatment with 5-ASA (Fig. S2F).

In conclusion, the ability of 5-ASA to check aerobic-respiration-dependent (Fig. S2F) E. coli expansion (Fig. 1C) correlated with a return of homeostatic functions of epithelial cells in DSS-treated mice, including restoration of epithelial hypoxia (Fig. 1E and F), and reestablished mitochondrial bioenergetics in the colonic epithelium (Fig. 1G and H). We thus wanted to determine whether 5-ASA directly activates PPAR-γ in colonic epithelial cells to check the growth of E. coli. Stimulation of cultured human colonic cancer epithelial (CaCo-2) cells with 5-ASA induced expression of PPARG and the PPAR-γ-activated gene ANGPTL4 (Fig. 2A) and triggered synthesis and the nuclear localization of PPAR-γ (Fig. 2B). Although there was no significant effect on either basal or maximal mitochondrial respiration (data not shown), 5-ASA stimulation increased the spare mitochondrial respiratory capacity in CaCo-2 cells (Fig. 2C), which was indicative of increased mitochondrial bioenergetics in response to cellular stress (i.e., inflammation).

5-ASA activates epithelial PPAR-γ signaling to check the growth of E. coli during colitis.

To investigate the importance of epithelial PPAR-γ signaling as a target for 5-ASA therapy in vivo, we generated mice lacking PPAR-γ specifically in the intestinal epithelium (Pparg^fl/fl Villin^cre/– mice) along with wild-type littermate control animals (Pparg^fl/fl Villin^−/− mice) using mice obtained from The Jackson Laboratory that were confirmed to be Enterobacterales free. Mice were engrafted with indicator strains for assessing the bioavailability of respiratory electron acceptors in the colon (i.e., a 1:1 mixture of E. coli strain Nissle 1917 and an isogenic cydA napA narG narZ mutant). Consistent with a previous report (21), mice lacking epithelial PPAR-γ signaling were more susceptible to DSS-induced colitis, as indicated by a worsened shortening of the colon (Fig. 3A) and elevated histopathology scoring (Fig. 3B and C). Notably, 5-ASA treatment ameliorated signs of inflammation in DSS-treated littermate control mice, but not in DSS-treated mice lacking epithelial PPAR-γ signaling (Fig. 3A to D), pointing to the colonic epithelium as a key player in negotiating the anti-inflammatory activity of 5-ASA.

Importantly, 5-ASA treatment blunted DSS-induced E. coli expansion in the colon only in littermate control mice, not in mice lacking epithelial PPAR-γ signaling (Fig. 4A). These data did not support the idea that 5-ASA is a direct inhibitor of E. coli growth in vivo (24) but instead suggested that the drug checks the growth of Enterobacterales indirectly by activating PPAR-γ signaling in the host epithelium. Further analysis of the consequences of epithelial PPAR-γ signaling revealed that 5-ASA treatment restored epithelial hypoxia in DSS-treated littermate control mice but not in DSS-treated mice lacking epithelial PPAR-γ signaling (Fig. 4B and C), suggesting that 5-ASA acts on the epithelium to restore mitochondrial bioenergetics. Consistently with this idea, colonic epithelial ATP levels and PDH levels were reinstated by 5-ASA supplementation in DSS-treated littermate control mice but not in DSS-treated mice lacking epithelial PPAR-γ signaling (Fig. 4D and Fig. S3A). Finally, 5-ASA supplementation diminished the respiration-dependent growth advantage of wild-type E. coli over the growth of a cydA napA narG narZ mutant in DSS-treated littermate control mice but not in DSS-treated mice lacking epithelial Pparg expression (Fig. 4E), which was consistent with the proposed role of epithelial PPAR-γ signaling in limiting the availability of respiratory electron acceptors in the colonic lumen to check the growth of facultative anaerobic Enterobacterales (29).

Contact for reagent and resource sharing.

Further information and requests for resources and reagents should be directed to the lead contact, Andreas J. Bäumler.

Experimental model and subject details. (i) Bacterial strains and culture conditions.

E. coli Nissle 1917 is a commensal human isolate (43) that has been marketed as a probiotic. The E. coli Nissle 1917 cydA napA narG narZ mutant and the E. coli Nissle 1917 cydA mutant used in this study have been described previously (29). E. coli strains were routinely grown aerobically at 37°C in LB broth (BD Biosciences) or on LB agar plates. When appropriate, antibiotics were added to the medium at the following concentrations: 0.1 mg/ml carbenicillin and 0.05 mg/ml kanamycin. For competitive infection experiments, strains were grown in a hypoxia chamber (0.8% oxygen) at 37°C in LB broth (BD Biosciences).

(ii) Caco-2 cell culture.

Caco-2 cells (ATCC; HTB-37) were cultured in minimal essential medium (MEM) (Gibco; 11090099) supplemented with 10% fetal bovine serum (FBS) (Gibco; 16140071), 1% GlutaMAX-I (Gibco; 35050061), 1% MEM nonessential amino acids (Gibco; 11140-030), and 1 mM sodium pyruvate (Gibco; 11360-070).

(iii) Animal experiments.

All experiments in this study were approved by the Institutional Animal Care and Use Committee at the University of California Davis. Female and male C57BL/6J mice, aged 8 to 10 weeks, were obtained from The Jackson Laboratory. Female and male C57BL/6 Pparg^fl/fl Villin^cre– and littermate Pparg^fl/fl Villin^−/− (control) mice were generated at UC Davis by mating Pparg^fl/fl mice with Villin^cre/– mice (The Jackson Laboratory).

(a) DSS treatment. Male and female C57BL/6J mice were given 3% DSS (Alfa Aesar) in their drinking water continuously for 8 days. At day 4 of DSS treatment, mice were inoculated with 1 × 10⁹ CFU of a 1:1 mixture of the above-indicated E. coli strains. Samples were collected at 4 days postinfection.

(b) 5-ASA treatment. Male C57BL/6J mice were given chow (Envigo, Taklad) supplemented with 2.5 g 5-ASA (Sigma-Aldrich)/kg of body weight for 9 to 10 days. At day 3 of 5-ASA supplementation, mice were given 2.5% DSS in their drinking water for the remainder of the experiment. At day 4 of DSS treatment, mice were inoculated with 1 × 10⁹ CFU of a 1:1 mixture of the above-indicated E. coli strains. Samples were collected either 2 or 3 days after infection.

(c) Experiments with Pparg^fl/fl Villin^cre/− mice. Female and male Pparg^fl/fl Villin^cre/− mice and littermate Pparg^fl/fl Villin^−/− control mice, aged 8 to 14 weeks, were given chow supplemented with 2.5 g/kg 5-ASA for 9 days. At day 3 of supplementation, mice were given 2.5% in their drinking water for the remainder of the experiment. At day 4 of DSS treatment, mice were inoculated with 1 × 10⁹ CFU of a 1:1 mixture of the above-indicated E. coli strains. Samples were collected at 2 days after infection.

Method details. (i) Colonocyte isolation.

The colon and part of the cecum were opened lengthwise and cut into 2- to 4-cm pieces, collected in 15 ml of ice-cold 1× RPMI 1640 buffer (Gibco) in a 50-ml Falcon tube, and cleaned with 20 ml of ice-cold 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco) in another 50-ml Falcon tube. The tissue was then placed into 15-ml conical centrifuge tubes filled with 10 ml of ice-cold dissociation reagent 1 (30 mM EDTA, 1.5 mM dithiothreitol [DTT], diluted into 1× DPBS) and placed on ice for 20 min. Tissues were then placed into a 15-ml conical centrifuge tube filled with 6 ml of warm (37°C) dissociation reagent 2 (30 mM EDTA, diluted into 1× DPBS) and incubated for 10 min at 37°C. After this incubation, tubes were shaken vigorously for 30 s to detach the epithelium from the basement membrane, for a total of about 80 to 90 shake cycles. Remnant intestinal tissue was removed, and the pellet cell solution was centrifuged at 800 × g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in 1 ml of Tri reagent (Molecular Research Center) for subsequent RNA extraction or in radioimmunoprecipitation assay (RIPA) buffer for metabolism analysis.

(ii) ATP measurements.

For measuring intracellular ATP levels, primary colonocytes were isolated as described above and then deproteinized by using a deproteinizing sample preparation kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Lactate measurements in colonocyte lysates were performed by using an ATP colorimetry assay kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions.

(iii) PDH activity measurements.

To measure PDH activity, primary colonocytes were isolated as described above and then lysed with RIPA buffer, and cellular debris were removed by centrifugation for 5 min at 13,000 rpm at room temperature. PDH activity was measured with supernatants by using the PDH activity assay kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions.

(iv) Histopathological analysis.

Colonic tissues were fixed in 10% phosphate-buffered formalin, and 5-μm sections of the tissue samples were stained with hematoxylin and eosin (H&E). Representative images were taken using an Olympus BX41 microscope at a magnification of ×10. Scoring of blinded tissue sections was performed by a veterinary pathologist based on the criteria listed in Table S2 in the supplemental material.

(v) Alcian blue staining.

Colon and cecal tissues were fixed in 10% phosphate-buffered formalin, and 5-μm sections of the tissue samples were stained with alcian blue. Pictures were captured on an AxioCam camera (2.2-μm by 2.2-μm pixel distance). Quantification of mature mucus-producing, alcian blue-positive cells was conducted based on a comet-like feature of the cells, with dense blue staining on the apical side of colonic crypt within the frame. Each measurement was conducted based on 5 frames per individual mouse; the average for each group was based on data from six mice per group determined at a magnification of ×40.

(vi) Hypoxia staining.

For detection of hypoxia, mice were treated with 60 mg/kg of pimonidazole HCl intraperitoneally (Hypoxyprobe-1 kit; Hypoxyprobe) 1 h prior to euthanasia. Colon samples were fixed in 10% phosphate-buffered formalin, and paraffin-embedded tissue was blocked with mouse-on-mouse blocking reagent (Vector Labs) and probed with mouse anti-pimonidazole monoclonal IgG1 (monoclonal antibody 4.3.11.3). Then, slides were stained with Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories). Samples were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) using SlowFade Gold mountant. Samples were scored based on the degree of colonic epithelial hypoxia (0, no hypoxia; 1, mild focal hypoxia; 2, moderate multifocal hypoxia; 3, intense diffuse hypoxia). Representative images were obtained using a Zeiss Axiovert 200 M fluorescence microscope and were brightness adjusted.

(vii) Fluorescence microscopy.

Caco-2 cells were cultured in T25 flasks as described previously. After reaching confluence, Caco-2 cells were harvested from each flask and resuspended in 15 ml MEM. Autoclaved glass coverslips were placed into a 24-well plate, and 500 μl of Caco-2 cell suspension was added to each well. After 48 h of incubation, Caco-2 cells were treated with 15 mM 5-ASA (Acros Organics) for 24 h. Cells were then washed 1× in PBS and permeabilized with 0.2% Triton X-100 for 2 min on ice. Permeabilized cells were treated with 4% paraformaldehyde for 20 min at room temperature and then washed with PBS. Ammonium chloride (NH₄Cl) was then added to cells for 15 min. Afterwards, cells were treated with 0.05% Triton X-100 for 10 min on ice. The surface was blocked by 10% goat serum with 0.3 M glycine for 1 h at room temperature. Subsequently, the primary anti-PPAR-γ antibody (1:100; Santa Cruz Biotechnology) was incubated overnight at 4°C. The secondary antibody (1:1,000; BD Pharmingen) was then incubated for 1 h at room temperature. Finally, the cells were incubated with phalloidin for labeling actin filaments. Between each step, the cells were washed in PBS. The stained cells were imaged using a Zeiss Axioplan 2 microscope with DAPI-FITC (fluorescein isothiocyanate)-TRITC (tetramethyl rhodamine isocyanate) excitation filter sets. Images were analyzed using ImageJ software.

(viii) Caco-2 RNA extraction and quantitative reverse transcription-PCR.

For gene expression assays, Caco-2 cells were grown until they were 70% confluent and then seeded into 6-well plates. After 48 h, 15 mM 5-ASA (Acros Organics) was added to cells and incubated for 6 h. Cells were then washed with DPBS, 0.05% trypsin-EDTA (Gibco) was added, and cells were harvested. Cell pellets were stored at −80°C. RNA was isolated according to the protocol provided (Norgen Biotek Corporation) and stored at −80°C. RNA was reverse transcribed using an iScript gDNA Clear cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using SYBR green (SsoAdvanced; Bio-Rad) for PPAR-γ and ANGPTL4. Primers are listed in Table S1. The expression of target genes was normalized to that of 18S rRNA.

(ix) Caco-2 cell measurement of oxygen consumption rates.

Experiments were performed using the Seahorse XF Cell Mito stress test kit (Agilent; 103708-100) according to the manufacturer’s instructions and carried out using an Agilent Seahorse XFe96 analyzer. Two days prior to 5-ASA treatment, Caco-2 cells were seeded in an XF cell culture microplate (Agilent) and placed in a tissue culture incubator at 37°C. One day prior to the assay, a utility plate (Agilent) was hydrated overnight with tissue culture-grade water at 37°C in a non-CO₂ incubator. Immediately prior to the running of the assay, water was removed from the utility plate, replaced with 200 μl of prewarmed 37°C XF calibrant (Agilent), and placed in a non-CO₂ incubator for 45 min to 1 h. Also immediately prior to the running of the assay, spent culture medium was replaced with 180 μl Seahorse XF Dulbecco’s modified Eagle’s medium (DMEM), pH 7.4 (Agilent; 103575-100) supplemented with 5.5 mM glucose (Agilent; 103577-100), 1% GlutaMAX-I, and 1 mM sodium pyruvate (Agilent complete medium), and cells were placed in a non-CO₂ incubator at 37°C for 1 h. Stock compounds were prepared and loaded into the sensor cartridge (Agilent) ports according to the manufacturer’s instructions, with maintenance of a final well concentration of 1.0 μM oligomycin, 0.25 μM FCCP {2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile}, and 0.5 μM rotenone/antimycin A.

(x) Bacterial growth assay.

To determine how 5-ASA affects the growth of E. coli Nissle 1917 in vitro, LB broth was prepared with increasing amounts of 5-ASA. 5-ASA (Sigma-Aldrich) was dissolved directly in LB broth at a final concentration of 2 mg/ml. Sodium hydroxide (Honeywell) was added to the medium until 5-ASA dissolved. LB broth containing 2 mg/ml 5-ASA was diluted in LB broth to final 5-ASA concentrations of 1 mg/ml and 0.25 mg/ml. The pH of LB broth alone and LB broth plus 5-ASA were matched to pH 7.0 using hydrochloric acid or sodium hydroxide and measured using pH strips. Overnight cultures of E. coli Nissle 1917 were harvested, washed in PBS, and added to a final optical density at 600 nm (OD₆₀₀) of 0.0001 in LB broth alone or LB broth containing 5-ASA. For anaerobic growth assays, the OD₆₀₀ was measured every hour for 24 h using Epoch 2 plate reader (BioTek) at 37°C with shaking. For aerobic growth assays, cultures were grown at 37°C with shaking and the OD₆₀₀ was measured at 4, 8, 12, and 24 h.

(xi) Quantification and statistical analysis.

To analyze ratios (i.e., competitive indices and fold changes in mRNA levels), bacterial numbers were transformed logarithmically prior to statistical analysis. An unpaired Student t test was used on the transformed data to determine whether differences between groups were statistically significant (P < 0.05). To determine the statistical significance of differences in total histopathology scores, a Mann-Whitney U test was used.