Trans-3-indoleacrylic acid ameliorates dextran sulfate sodium-induced colitis by modulating macrophage M1 polarization through the aryl hydrocarbon receptor pathway

Abstract

BACKGROUND

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease with a steadily increasing global incidence. Controlling mucosal inflammation represents a crucial therapeutic goal; however, safe and effective long-term treatments remain limited. Increasing evidence suggests that microbiota-derived tryptophan metabolites regulate intestinal immune homeostasis and participate in the pathogenesis of UC.

AIM

To investigate whether trans-3-indoleacrylic acid (IDA) alleviates dextran sulfate sodium (DSS)-induced colitis by regulating macrophage polarization.

METHODS

Male C57BL/6 mice with DSS-induced colitis and lipopolysaccharide/interferon-γ-stimulated THP-1-derived macrophages and bone marrow-derived macrophages were treated with IDA. Aryl hydrocarbon receptor (AHR) signaling involvement was evaluated using CH-223191-mediated pharmacological inhibition in vivo and siRNA-mediated AHR knockdown in vitro. Colonic inflammation, intestinal barrier integrity, and macrophage phenotypes were assessed by histological analysis, enzyme-linked immunosorbent assay, quantitative real-time PCR, Western blotting, and flow cytometry. Gut microbiota composition was characterized by 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) were quantified by targeted metabolomic analysis.

RESULTS

IDA treatment markedly alleviated colitis severity, preserved intestinal barrier integrity, and reduced pro-inflammatory mediators, including inducible nitric oxide synthase, interleukin-1β, and interleukin-6. Importantly, IDA suppressed M1 macrophage polarization without promoting M2 macrophage polarization. Mechanistically, IDA activated AHR signaling, as evidenced by increased expression of downstream targets, including CYP1A1 and CYP1B1, while AHR inhibition or knockdown attenuated the inhibitory effects of IDA on M1 macrophage polarization. Furthermore, IDA treatment was associated with alterations in gut microbiota composition, characterized by reduced abundance of potential pathobionts, such as Escherichia-Shigella, and increased abundance of beneficial commensals, including Akkermansia and Lactobacillus. Targeted metabolomic analysis revealed that IDA treatment was accompanied by increased intestinal levels of SCFAs, particularly acetic acid and butyric acid.

CONCLUSION

IDA alleviates DSS-induced colitis by suppressing M1 macrophage polarization through the AHR-CYP1A1 signaling pathway and is associated with alterations in gut microbiota composition and SCFA profiles.

Key Words: Ulcerative colitis; Trans-3-indoleacrylic acid; Macrophage polarization; Aryl hydrocarbon receptor; Gut microbiota; Short-chain fatty acids

Core Tip: Microbiota-derived tryptophan metabolites contribute to intestinal immune homeostasis, but the immunoregulatory role of trans-3-indoleacrylic acid (IDA) in ulcerative colitis (UC) remains unclear. This study demonstrates that IDA alleviates dextran sulfate sodium-induced colitis by suppressing pro-inflammatory M1 macrophage polarization through activation of the aryl hydrocarbon receptor (AHR)-CYP1A1 pathway. IDA treatment is also associated with alterations in gut microbiota composition and short-chain fatty acid profiles. These findings identify the IDA-AHR-macrophage axis as a potential therapeutic target for UC.

INTRODUCTION

Ulcerative colitis (UC), a chronic and relapsing inflammatory bowel disease (IBD), is clinically characterized by diarrhea, persistent abdominal pain, tenesmus, and rectal bleeding[1]. Due to its steadily increasing global incidence, UC imposes an increasing burden on health care systems[2]. Symptom relief is regarded as a short-term goal in current UC management, whereas endoscopic mucosal healing is considered a long-term therapeutic objective[3]. Studies have indicated that patients who achieve endoscopic mucosal healing have an improved prognosis, as evidenced by prolonged remission periods and lower hospitalization and surgery rates[4]. A balanced gut microbiota is essential for maintaining host immune and metabolic homeostasis. Dysbiosis impairs metabolic functions and disrupts the balance of immune cells, leading to persistent mucosal inflammation[5]. Therefore, targeting the interactions among the gut microbiota, microbial metabolites, and host immunity represents a critical strategy for achieving mucosal healing and improving the prognosis of UC patients.

Intestinal macrophages are strategically positioned at the host-environment interface to sense microbial and immunological cues within the gut. Under physiological conditions, these cells exhibit a highly regulated anti-inflammatory phenotype, which balances pathogen clearance and immune tolerance to the commensal microbiota[6,7]. During UC pathogenesis, however, this homeostasis is disrupted, resulting in macrophage polarization toward a proinflammatory phenotype, referred to as M1 macrophages. These cells accumulate extensively in inflamed colonic tissues and exacerbate mucosal damage by secreting inflammatory cytokines, disrupting tight junction (TJ) proteins, and inducing epithelial apoptosis[8]. Therefore, inhibiting the proinflammatory activity of M1 macrophages and promoting their transition toward an anti-inflammatory phenotype may improve the immune microenvironment in patients with UC.

Microbiota-derived metabolites act as critical mediators of host-microbe interactions and play essential roles in maintaining intestinal homeostasis[9]. Common metabolites include short-chain fatty acids (SCFAs), secondary bile acids, and tryptophan-derived metabolites. Among these metabolites, indole and indole derivatives, a major class of tryptophan metabolites, have attracted increasing attention. Specifically, dietary tryptophan is metabolized by commensal bacteria, such as Bifidobacterium and Lactobacillus, via the indole pathway to generate a range of metabolites, including indole-3-lactic acid (ILA), indole-3-acetic acid (IAA), and indole-3-propionic acid (IPA)[10,11]. In the intestinal microenvironment, indole derivatives function as endogenous aryl hydrocarbon receptor (AHR) ligands that regulate intestinal barrier integrity, mucosal immune homeostasis, and the gut microbiota composition[12]. However, compared with healthy controls, IBD patients exhibit significantly reduced colonic AHR expression[13]. In addition, impaired tryptophan metabolism has been observed in individuals with IBD, as reflected by decreased fecal levels of AHR ligands[14]. Notably, exogenous indole derivatives can ameliorate experimental colitis by regulating intestinal immune responses. For instance, indole-3-aldehyde (IAld) has been reported to activate AHR signaling, leading to increased expression of CYP1A1 and CYP1B1 and subsequent inhibition of M1 macrophage polarization, thus alleviating colitis[15]. This protective effect is reversed by the AHR inhibitor (AHRI) CH-223191, further supporting the critical role of the indole-AHR axis in regulating intestinal immune homeostasis.

Trans-3-indoleacrylic acid (IDA) is a bioactive metabolite derived from tryptophan metabolism by Bacteroides and Lactobacillus and exerts anti-inflammatory, antioxidant, and antifibrotic effects in various disease models[16,17]. Existing studies on the role of IDA in intestinal homeostasis have focused primarily on epithelial barrier function and intestinal epithelial cells. Jing et al[18] demonstrated that IDA is the key active component that mediates the protective effects of Wuji Wan on experimental colitis by promoting intestinal barrier regeneration through the induction of colonic IL-22 expression. Dong et al[19] reported that IDA inhibits the necroptosis of intestinal epithelial cells via the AHR-SOCS5 axis, thus alleviating necrotizing enterocolitis (NEC). With respect to immune regulation, IDA has been shown to mitigate intermittent fasting-induced intestinal inflammation by suppressing Th17 cell differentiation[20]. Despite the extensive benefits of IDA in maintaining intestinal homeostasis, its regulatory role in colonic macrophages and its detailed mechanisms in UC remain unclear.

In the present study, we investigated whether IDA exerts protective effects on DSS-induced colitis and elucidated its underlying molecular mechanisms. We first demonstrated that IDA alleviates intestinal inflammation and improves intestinal barrier function. Using pharmacological inhibition in vivo and siRNA-mediated AHR knockdown in vitro, we further showed that IDA suppresses M1 macrophage polarization via the AHR-CYP1A1 axis. Finally, 16S rRNA gene sequencing and a targeted metabolomic analysis revealed that IDA treatment reshaped the gut microbiota and altered SCFA profiles.

MATERIALS AND METHODS

Animal experiments

Male C57BL/6 mice (8 weeks old, 18-22 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. [Changsha, China; License No. of Laboratory Animal Use: SYXK (Xiang) 2020-0019]. All experimental procedures were approved by the Ethics Committee for Animal Experiments of Central South University (No. CSU-2022-0442). The mice were housed under specific pathogen-free conditions at 22 ± 2 °C with a 12-hour light/dark cycle and were allowed free access to sterilized chow and water. Following a 1-week acclimatization period, the mice were randomly assigned to different experimental groups. All mice exhibited comparable baseline body weights before treatment initiation.

Eighteen mice were randomly divided into the following 3 groups (n = 6 mice) to evaluate the protective effect of IDA on DSS-induced colitis: (1) The control group, which received a daily oral gavage of 0.5% sodium carboxymethyl cellulose (CMC-Na; Aladdin, Shanghai, China) vehicle for 14 consecutive days (day -7 to day 7), with normal drinking water; (2) The DSS group, which received a daily oral gavage of 0.5% CMC-Na vehicle for 14 days, with 2.5% (w/v) dextran sulfate sodium (DSS, MW 36-50 kDa; MP Biochemicals, Solon, OH, United States) supplemented in the drinking water from day 1 to day 7 to induce acute colitis; and (3) The DSS + IDA group, which received a daily oral gavage of IDA (50 mg/kg; MedChemExpress, Shanghai, China) dissolved in 0.5% CMC-Na for 14 days, along with 2.5% DSS in the drinking water from day 1 to day 7. The dose of IDA was set based on previously published in vivo studies[21-23]. The gavage volume was adjusted to 0.1 mL per 10 g body weight. Weight loss, stool consistency and the degree of intestinal bleeding were monitored daily to evaluate the disease activity index (DAI). On day 7, an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) was administered to induce anesthesia in all the mice. Euthanasia was then performed via cervical dislocation. Finally, fecal samples, peripheral blood, spleens, and colons were collected for subsequent assays. The sample size was determined based on prior experimental experience and animal welfare considerations. Six mice per group were considered sufficient to detect biologically meaningful differences while minimizing unnecessary animal use.

An independent experiment was conducted with another 18 mice that were randomly allocated into the following 3 groups (n = 6 mice) to further elucidate whether the protective effect of IDA depends on AHR: (1) The DSS group; (2) The DSS + IDA group; and (3) The DSS + IDA + AHRI group. Mice in the DSS + IDA + AHRI group were coadministered IDA (50 mg/kg) and the AHR-specific inhibitor CH-223191 (10 mg/kg; MedChemExpress, Shanghai, China), both dissolved in 0.5% CMC-Na, via daily oral gavage from day -7 to day 7. The DSS administration protocol (days 1-7), gavage volume, daily DAI monitoring, and sample collection procedures were the same as those described above. No unexpected adverse events or mortality occurred during the study.

Assessment of the DAI

Colitis severity was assessed daily using the DAI, a composite score based on three parameters: Weight loss, stool consistency, and the degree of intestinal bleeding. The scoring criteria were defined as follows: (1) Weight loss: 0 (none), 1 (1%-5%), 2 (6%-10%), 3 (11%-15%), and 4 (> 15%); (2) Stool consistency: 0 (normal), 2 (soft stools), and 4 (watery diarrhea); and (3) Intestinal bleeding: 0 (negative), 2 (positive occult blood), and 4 (gross rectal bleeding). The final DAI score was calculated as the sum of these three parameters, with a maximum score of 12.

Histological staining and evaluation

Distal colonic tissues were harvested, fixed with 10% neutral-buffered formalin for 24 hours, and embedded in paraffin. The tissue blocks were cut into 5-μm sections and stained with hematoxylin and eosin (H&E) to evaluate the overall tissue morphology, inflammatory cell infiltration, crypt structure, and epithelial integrity. Furthermore, alcian blue-periodic acid-Schiff (AB-PAS) staining of the tissue sections was performed to assess the number of goblet cells. The histopathological evaluation was independently performed by two experienced pathologists in a blinded manner. Histopathological scores were determined according to the criteria detailed in Supplementary Table 1, and goblet cell numbers were quantified using ImageJ/Fiji software (NIH, Bethesda, MD, United States).

Assessment of intestinal barrier permeability

Intestinal epithelial barrier permeability was evaluated using fluorescein isothiocyanate-dextran (FITC-Dextran; MedChemExpress, Monmouth Junction, NJ, United States). Briefly, the mice were fasted for 4 hours and subsequently administered FITC-dextran (600 mg/kg body weight) by oral gavage. Peripheral blood was collected 5 hours later, and serum was obtained following centrifugation at 3000 × g for 10 minutes at 4 °C. The fluorescence intensity of FITC-dextran in serum was quantified using a microplate reader (BioTek, Winooski, VT, United States) at excitation/emission wavelengths of 485/525 nm. Serum FITC-dextran concentrations were calculated from a standard curve prepared by serial dilution.

16S rRNA gene sequencing and analysis

Fresh fecal samples from the DSS and DSS + IDA groups were subjected to 16S rRNA gene sequencing to evaluate the effect of IDA on the gut microbiota. Genomic DNA was extracted using the E.Z.N.A. Soil DNA Kit (Omega Biotek, Norcross, GA, United States), and its quality and concentration were verified via agarose gel electrophoresis and a Qubit 4.0 fluorometer (Thermo Fisher Scientific), respectively. The bacterial 16S rRNA V3-V4 region was PCR-amplified using the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′)[24]. Amplicons were electrophoretically verified, purified using Hieff NG^TM DNA Selection Beads (Yeasen, China), quantified using a Qubit system, and pooled in equimolar amounts for Illumina paired-end library construction. Sangon Biotech Co., Ltd. (Shanghai, China) performed 2 × 250 bp paired-end sequencing on an Illumina MiSeq platform (Illumina, San Diego, CA, United States).

Paired-end reads were merged via PEAR (v 0.9.8)[25], and operational taxonomic units (OTUs) were clustered at 97% similarity using USEARCH (v 11.0.667). After chimeric and singleton sequence removal, representative sequences were taxonomically mapped by comparison with the Ribosomal Database Project database[26]. The microbiota profiles were subsequently evaluated. Alpha diversity was estimated using the Chao1 and Shannon indices, while intergroup differences in beta diversity were visualized via principal coordinate analysis (PCoA). Linear discriminant analysis (LDA) effect size was employed to identify microbial biomarkers with a LDA score threshold of > 4.0.

Quantification of SCFAs

SCFAs in the cecal contents were analyzed by gas chromatography-tandem mass spectrometry using an Agilent 8890 GC coupled with a 7000D mass spectrometer (Agilent Technologies, Santa Clara, CA, United States). Approximately 20 mg of sample was placed in a 2 mL tube, followed by the addition of 1 mL of 0.5% (v/v) phosphoric acid and a steel bead. After homogenization, the mixture was vortexed for 10 minutes and sonicated for 5 minutes. The extract was then centrifuged at 12000 rpm for 10 minutes at 4 °C, after which 100 μL of the supernatant was collected. MTBE (500 μL) containing internal standards (CNW Technologies, Düsseldorf, Germany) was added, and the mixture was vortexed for 3 minutes and sonicated for 5 minutes, followed by centrifugation under the same conditions. The upper organic phase was collected for subsequent analysis.

Chromatographic separation was performed on a DB-FFAP column (30 m × 0.25 mm × 0.25 μm; J&W Scientific, Folsom, CA, United States) with helium as the carrier gas at a rate of 1.2 mL/minute. A 1 μL aliquot was injected at a split ratio of 5:1. The oven temperature was programmed from 50 °C (2 minutes hold) to 220 °C at a rate of 40 °C/minute. The injector inlet and transfer line temperatures were both set at 230 °C. Detection was conducted in multiple reaction monitoring mode.

Cell culture and treatments

The human monocytic cell line THP-1 was acquired from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China) and cultured in RPMI 1640 medium (Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. THP-1 cells were treated with phorbol 12-myristate 13-acetate (100 ng/mL; MedChemExpress, Monmouth Junction, NJ, United States) for 24 hours to induce macrophage differentiation. The differentiated cells were then stimulated with 100 ng/mL lipopolysaccharide [LPS; Escherichia coli (E. coli) O55: B5; Sigma-Aldrich, St. Louis, MO, United States] and 20 ng/mL human interferon-γ (IFN-γ; PeproTech, Cranbury, NJ, United States) for 24 hours to induce M1 polarization.

Bone marrow-derived macrophages (BMDMs) were obtained from the femurs and tibias of male C57BL/6 mice. After aseptic dissection, the bone marrow was flushed with ice-cold sterile phosphate-buffered saline (PBS), passed through a 70-μm cell strainer, and centrifuged at 500 × g for 5 minutes at 4 °C. The cell suspension was cultured in DMEM (Gibco, Grand Island, NY, United States) supplemented with 10% FBS, 1% penicillin-streptomycin, and macrophage colony-stimulating factor (25 ng/mL; PeproTech, Cranbury, NJ, United States). The cells were maintained for 7 days to induce macrophage differentiation, and the medium was replaced every 2 days. Mature BMDMs were then stimulated with LPS (100 ng/mL) and murine IFN-γ (50 ng/mL; PeproTech, Cranbury, NJ, United States) for 24 hours to induce M1 polarization.

THP-1-derived macrophages (hereafter referred to as THP-1 macrophages) and BMDMs were used to evaluate the anti-inflammatory effects of IDA and the involvement of AHR signaling. Cells were divided into four groups: (1) The control group; (2) The LPS + IFN-γ group; (3) The IDA + LPS + IFN-γ group (IDA group); and (4) The IDA + LPS + IFN-γ + siAHR group (siAHR group). For AHR knockdown experiments, cells were transfected with AHR-specific siRNA or corresponding negative control siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. Twenty-four hours after transfection, AHR knockdown efficiency was verified by Western blot analysis before subsequent functional experiments. The sequences of AHR-specific siRNAs targeting human and mouse AHR are provided in Supplementary Tables 2 and 3, respectively. After confirmation of AHR knockdown, cells were pretreated with IDA (100 μM) for 12 hours and subsequently stimulated with LPS and IFN-γ for an additional 24 hours. Cells and culture supernatants were collected for subsequent analyses.

Cell viability assay

Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, Shanghai, China). THP-1 and BMDMs were seeded in 96-well plates and allowed to adhere and differentiate, as described above. After differentiation, the cells were treated with the indicated concentrations of IDA (0 μM, 1 μM, 10 μM, 50 μM, 100 μM, and 500 μM) for 36 hours. Subsequently, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 hours at 37 °C in the dark. Cell viability was determined by measuring the absorbance (OD) at 450 nm using a microplate reader (BioTek, Winooski, VT, United States).

Flow cytometry analysis

Flow cytometry was performed on single-cell suspensions prepared from mouse colonic lamina propria mononuclear cells, THP-1 macrophages, and BMDMs to assess the polarization of M1 macrophages. For colonic tissue, the samples were collected, minced, enzymatically digested, and filtered through a 70-μm nylon cell strainer to generate single-cell suspensions. THP-1 macrophages and BMDMs were harvested and washed with ice-cold PBS (Servicebio, Wuhan, China). The cells were preincubated with species-specific Fc receptor blocking reagents (BioLegend, San Diego, CA, United States) for 15 minutes at 4 °C to minimize nonspecific binding.

The cells were then stained with fluorophore-conjugated monoclonal antibodies for 30 minutes at 4 °C in the dark. After surface staining, the cells were fixed and permeabilized using a fixation/permeabilization kit (Elabscience, Wuhan, China) according to the manufacturer’s instructions for CD206 staining. For the colonic tissue samples, the cells were stained with Alexa Fluor 488 (AF488)-conjugated anti-mouse F4/80 (Cat No. E-AB-F0995 L), PerCP-Cy5.5-conjugated anti-mouse CD11b (Cat No. E-AB-F1081J), PE-conjugated anti-mouse CD86 (Cat No. E-AB-F0994D), and APC-conjugated anti-mouse CD206 (Cat No. E-AB-F1135E), all of which were purchased from Elabscience (Wuhan, China). THP-1 macrophages were stained with FITC-conjugated anti-human CD80 (Cat No. E-AB-F0992UC; Elabscience, Wuhan, China), PE-conjugated anti-human CD86 (Cat No. 374205; BioLegend, San Diego, CA, United States), and APC-conjugated anti-human CD206 (Cat No. E-AB-F1161E; Elabscience, Wuhan, China). BMDMs were stained with FITC-conjugated anti-mouse CD80 (Cat No. E-AB-F0992C), PE-conjugated anti-mouse CD86 (Cat No. E-AB-F0994D), and APC-conjugated anti-mouse CD206 (Cat No. E-AB-F1135E), all of which were purchased from Elabscience (Wuhan, China). After staining, the cells were washed and resuspended in staining buffer (Elabscience, Wuhan, China). Data were acquired on a BD FACSymphony A1 flow cytometer and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, United States).

Quantitative real-time PCR

Total RNA was extracted from the colonic tissues and cultured cells using TRIzol reagent (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. The concentration and purity of the extracted RNA were subsequently evaluated to confirm the quality of the samples. For cDNA synthesis, 1 μg of total RNA was reverse transcribed using a reverse transcription kit (Thermo Fisher Scientific, MA, United States). Quantitative PCR (qPCR) was performed on a LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) with SYBR Green qPCR Mix (TransGen Biotech, Beijing, China). The amplification protocol included an initial denaturation step at 94 °C for 30 seconds, followed by 40 cycles of 94 °C for 5 seconds and 60 °C for 30 seconds. The primer sequences are provided in Supplementary Tables 4 and 5. Relative gene expression was calculated using the 2^-ΔΔCt method, with β-actin used as the internal control.

Western blotting

Total protein was extracted from the colonic tissues and cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors. The protein concentration was determined using a BCA protein assay kit (Abbkine, Woburn, MA, United States). Equal amounts of protein were mixed with loading buffer (Bio-Rad, Hercules, CA, United States), denatured at 95 °C for 5 minutes, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States).

The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST; Servicebio, Wuhan, China) for 1 hour at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies against ZO-1 (1:2000; Cat No. 21773-1-AP), Occludin (1:5000; Cat No. 27260-1-AP), AHR monoclonal antibody (1:5000; Cat No. 67785-1-Ig), CYP1A1 (1:1000; Cat No. 13241-1-AP), and GAPDH (1:50000; Cat No. 60004-1-Ig). The membranes were subsequently washed four times with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:20000; Cat No. SA00001-2) or goat anti-mouse (1:20000; Cat No. SA00001-1) secondary antibodies for 1 hour at room temperature. All the antibodies mentioned above were purchased from Proteintech (Rosemont, IL, United States). After four additional washes with TBST, immunoreactive bands were detected using enhanced chemiluminescence reagent (Abbkine, Woburn, MA, United States), and band intensities were quantified using ImageJ/Fiji software (NIH, Bethesda, MD, United States).

Quantification of proinflammatory mediators

Specific enzyme-linked immunosorbent assay (ELISA) kits (Elabscience, Wuhan, China) were used to evaluate the inflammatory response by measuring iNOS, IL-1β, and IL-6 concentrations in colonic tissue homogenates and cell culture supernatants. The OD values were measured at 450 nm with a BioTek microplate reader (Winooski, VT, United States), and precise levels of inflammatory mediators were subsequently deduced based on interpolation from standard curves.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, United States). The data are presented as the mean ± SD. Differences among multiple groups were evaluated using one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. For 16S rRNA gene sequencing and metabolomic data, differences between two independent groups were evaluated using the Mann-Whitney test. For all in vivo experiments, each mouse was considered as an independent experimental unit. Statistical significance was defined as a P value < 0.05.

RESULTS

IDA ameliorates acute DSS-induced colitis in mice

We explored the protective effect of IDA in mitigating intestinal inflammation by establishing a mouse model of acute DSS-induced colitis. Before treatment initiation, all mice showed similar body weights. The mice were pretreated with IDA for 7 days, followed by the administration of 2.5% DSS in the drinking water for 7 days. Throughout the 14-day protocol, the mice in the IDA treatment group received 50 mg/kg IDA via oral gavage daily (Figure 1A). As expected, compared with the control treatment, DSS treatment induced significant body weight loss and elevated DAI scores, indicating the successful establishment of the model. However, IDA treatment markedly attenuated the weight loss and reduced the DAI score (Figure 1B and C). Given that DSS-induced colitis is typically characterized by colon shortening and splenomegaly, we measured the colon lengths of the mice and determined the spleen index (spleen weight/body weight) to assess the spleen alterations in mice with colitis. Compared with the DSS group, the IDA-treated group exhibited less colon shortening and significantly lower spleen index scores (Figure 1D-F). H&E staining of the distal colon tissue revealed that DSS administration induced notable inflammatory cell infiltration, crypt distortion or loss, and epithelial cell shedding. Conversely, these pathological changes were reversed in the IDA group, as evidenced by a marked reduction in the histological score (Figure 1G and H). Taken together, these results indicate that IDA significantly attenuates the clinical and pathological severity of DSS-induced colitis in mice.

Figure 1 Trans-3-indoleacrylic acid ameliorates acute dextran sulfate sodium-induced colitis in mice. A: Schematic of the animal experiment; B: Body weight change (%) of mice during dextran sulfate sodium (DSS) treatment; C: Disease activity index score during DSS treatment; D: Representative images of colons from each group; E: Colon length; F: Spleen index; G: Representative images of colonic sections stained with hematoxylin and eosin; H: Histological scores. The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. In B and C, the P values indicate the differences between groups on day 7. ^aP < 0.001, ^bP < 0.0001. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; DAI: Disease activity index; H&E: Hematoxylin and eosin.

IDA preserves intestinal barrier integrity and reduces intestinal permeability in mice with colitis

Given that the development of colitis is often accompanied by increased intestinal permeability and barrier dysfunction, we next investigated whether IDA could improve these parameters. Intestinal permeability was assessed via a FITC-dextran assay, and the expression levels of TJ proteins, including ZO-1, Occludin, and Claudin-1, were determined using quantitative real-time PCR (RT-qPCR) and Western blotting. Additionally, AB-PAS staining was performed to evaluate goblet cell abundance and mucin secretion. The data revealed a significant increase in serum FITC-dextran levels in the DSS group; however, IDA administration markedly attenuated this increase (Figure 2A). Furthermore, compared with DSS-treated mice, IDA-treated mice exhibited preserved TJ protein expression, suggesting that IDA exerts a protective effect on the physical intestinal barrier (Figure 2B-G). AB-PAS staining revealed a significant loss of goblet cells and a concomitant reduction in acidic mucin secretion in the DSS group, both of which were substantially preserved by IDA treatment (Figure 2H and I). Collectively, these results demonstrate that IDA effectively reduced intestinal permeability and bolstered barrier function by promoting TJ protein expression and mucin production.

Figure 2 Trans-3-indoleacrylic acid preserves intestinal barrier integrity and reduces intestinal permeability in mice with colitis. A: Fluorescence intensity of fluorescein isothiocyanate-dextran in serum; B-D: Relative mRNA expression of Tjp1, Ocln, and Cldn1 in colonic tissues; E-G: Representative immunoblots and the relative protein expression of ZO-1 and Occludin in colonic tissues; H: Representative images of colonic sections stained with alcian blue-periodic acid-Schiff (scale bars = 50 μm); I: Number of goblet cells. The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. ^aP < 0.01, ^bP < 0.001, ^cP < 0.0001. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; FITC-Dextran: Fluorescein isothiocyanate-Dextran; AB-PAS: Alcian blue-periodic acid-Schiff.

IDA activates AHR signaling and suppresses M1 macrophage polarization in vivo

The pathogenesis of colonic inflammation is characterized by the overproduction of inflammatory cytokines and the dysregulated activation of immune cells. Therefore, we further investigated the regulatory effects of IDA on the immune microenvironment. The concentrations of inflammatory mediators, including iNOS, IL-1β, and IL-6, in the colonic tissue were quantified using ELISA. Compared with control mice, DSS-induced mice exhibited elevated concentrations of these cytokines, whereas IDA administration significantly reduced their levels (Figure 3A-C). Consistently, RT-qPCR analysis of Nos2, Il1b, and Il6 mRNA expression revealed similar trends (Figure 3D-F).

Figure 3 Trans-3-indoleacrylic acid activates aryl hydrocarbon receptor signaling and suppresses M1 macrophage polarization in vivo. A-C: Concentration of iNOS, IL-1β, and IL-6 in colonic tissues; D-F: Relative mRNA expression of Nos2, Il1b, and Il6 in colonic tissues; G: Representative flow cytometry plots of macrophages from the colon lamina propria. Cells were pre-gated on the CD11b⁺ population. The upper right quadrant (Q2) indicates F4/80⁺CD86⁺ macrophages (M1 macrophages); H: Quantitative analysis of the percentage of F4/80⁺CD86⁺ macrophages among CD11b⁺ cells; I-K: Representative immunoblots and the relative protein expression of AHR and CYP1A1 in colonic tissues; L-O: Relative mRNA expression of Ahr, Cyp1a1, Cyp1b1, and Il22 in colonic tissues. The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. ^aP < 0.01, ^bP < 0.001, ^cP < 0.0001. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; AHR: Aryl hydrocarbon receptor.

Because macrophage polarization plays a critical role in regulating intestinal inflammation, we next investigated whether IDA affects macrophage polarization. Flow cytometry was performed to characterize macrophage subsets in the colonic lamina propria, with F4/80⁺CD86⁺ cells defined as M1 macrophages and F4/80⁺CD206⁺ cells defined as M2 macrophages. DSS administration significantly increased the proportion of F4/80⁺CD86⁺ M1 macrophages, whereas IDA treatment markedly reduced this population (Figure 3G and H). In contrast, the proportion of F4/80⁺CD206⁺ M2 macrophages showed no significant differences among the experimental groups (Supplementary Figure 1A). To further determine whether IDA regulates M2 macrophage polarization, the expression levels of M2-associated markers, including Mrc1, Arg1, and Chil3, were examined by RT-qPCR. Compared with DSS treatment alone, IDA administration did not significantly alter the expression levels of these M2-associated genes (Supplementary Figure 1B-D). These findings indicate that IDA selectively suppresses proinflammatory M1 macrophage polarization without promoting M2 macrophage differentiation, suggesting that the anti-inflammatory effects of IDA are primarily mediated through the inhibition of M1 macrophage activation rather than the induction of an M2 phenotype.

Given that IDA is a classic endogenous ligand of AHR, we further evaluated its effects on AHR signaling in the colonic tissue. Consistent with our expectations, the protein levels of AHR and CYP1A1, as well as the mRNA expression levels of Ahr, Cyp1a1, Cyp1b1, and Il22, were significantly upregulated in IDA-treated mice, suggesting that IDA effectively activated the AHR signaling pathway (Figure 3I-O). Taken together, these findings indicate that IDA suppresses M1 macrophage polarization while activating AHR signaling in vivo.

AHR inhibition abrogates the protective effects of IDA on DSS-induced colitis

To determine whether the protective effect of IDA was mediated by the AHR signaling pathway, DSS-induced mice were treated with the specific AHRI CH-223191. All mice exhibited comparable body weights and normal activity before the indicated interventions. Throughout the 14-day experimental regimen, the mice in the AHRI group received 50 mg/kg IDA and 10 mg/kg CH-223191 daily by oral gavage (Figure 4A). Consistent with our expectations, IDA administration mitigated body weight loss, DAI scores, colon shortening, splenomegaly, and histological damage in the mice with DSS-induced colitis (Figure 4B-H). Notably, pharmacological blockade of AHR via CH-223191 largely abrogated the beneficial effects of IDA on these parameters. Additionally, AB-PAS staining revealed that compared with the IDA group, the AHRI group exhibited decreased goblet cell abundance and reduced mucin secretion (Figure 4I-J), indicating that AHR inhibition effectively compromised the IDA-mediated restoration of the intestinal mucosal barrier. Collectively, these findings indicate that the pharmacological inhibition of AHR markedly attenuates the protective effects of IDA on DSS-induced colitis, supporting the involvement of AHR signaling in vivo.

Figure 4 Aryl hydrocarbon receptor inhibition abrogates the protective effects of trans-3-indoleacrylic acid on dextran sulfate sodium-induced colitis. A: Schematic of the animal experiment; B: Body weight change (%) of mice during dextran sulfate sodium (DSS) treatment; C: Disease activity index score during DSS treatment; D: Representative images of colons from each group; E: Colon length; F: Spleen index; G: Representative images of colonic sections stained with hematoxylin and eosin; H: Histological scores; I: Representative images of colonic sections stained with alcian blue-periodic acid-Schiff; J: Number of goblet cells. The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. In B and C, the P values indicate the differences between groups on day 7. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; AHRI: Aryl hydrocarbon receptor inhibitor; DAI: Disease activity index; H&E: Hematoxylin and eosin. AB-PAS: Alcian blue-periodic acid-Schiff.

IDA suppresses M1 macrophage polarization via AHR activation in vivo

To determine whether the immunomodulatory effects of IDA were mediated by AHR signaling, we quantified the levels of inflammatory mediators and the proportions of M1 macrophages in colonic tissues. ELISA and RT-qPCR revealed that the IDA intervention effectively reduced the protein and transcript levels of inflammatory mediators (including iNOS/Nos2, IL-1β/Il1b, and IL-6/Il6). However, these inhibitory effects were largely abrogated by CH-223191 treatment (Figure 5A-F). Furthermore, flow cytometry was performed to determine the proportion of colonic M1 macrophages. As shown in Figure 5G and H, IDA treatment significantly decreased the proportion of M1 macrophages, whereas CH-223191 treatment partially reversed this effect.

Figure 5 Trans-3-indoleacrylic acid suppresses M1 macrophage polarization via aryl hydrocarbon receptor activation in vivo. A-C: Concentration of iNOS, IL-1β, and IL-6 in colonic tissues; D-F: Relative mRNA expression of Nos2, Il1b, and Il6 in colonic tissues; G: Representative flow cytometry plots of macrophages from the colon lamina propria. Cells were pre-gated on the CD11b⁺ population. The upper right quadrant (Q2) indicates F4/80⁺CD86⁺ macrophages (M1 macrophages); H: Quantitative analysis of the percentage of F4/80⁺CD86⁺ macrophages among CD11b⁺ cells; I-K: Representative immunoblots and the relative protein expression of aryl hydrocarbon receptor and CYP1A1 in colonic tissues; L-O: Relative mRNA expression of Ahr, Cyp1a1, Cyp1b1, and Il22 in colonic tissues. The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; AHRI: Aryl hydrocarbon receptor inhibitor; AHR: Aryl hydrocarbon receptor.

We further validated the pharmacological antagonism of the AHR signaling pathway by assessing the expression levels of AHR, CYP1A1, CYP1B1 and IL-22. The results showed that CH-223191 treatment effectively reversed the IDA-induced upregulation of AHR-related molecules (Figure 5I-O). Taken together, these findings show that the pharmacological inhibition of AHR largely abolishes the suppressive effects of IDA on inflammatory mediator production and M1 macrophage polarization in vivo.

IDA attenuates LPS-stimulated M1 polarization through AHR signaling in vitro

An in vitro inflammatory model was established by stimulating THP-1 macrophages and BMDMs with LPS and IFN-γ to further investigate whether IDA exerts its anti-inflammatory effects through macrophage-intrinsic AHR signaling. Based on the results of the cell viability assay and RT-qPCR analyses, 100 μM IDA was selected as the working concentration for subsequent experiments (Supplementary Figure 2). AHR knockdown efficiency was first confirmed by Western blot analysis before functional experiments were performed (Figure 6A and Supplementary Figure 3A).

Figure 6 Trans-3-indoleacrylic acid attenuates lipopolysaccharide-stimulated M1 polarization through aryl hydrocarbon receptor signaling in vitro. A: Representative immunoblots of aryl hydrocarbon receptor (AHR) in AHR knockdown THP-1 macrophages; B-D: Concentration of iNOS, IL-1β, and IL-6 in THP-1 macrophage culture supernatants; E-G: Relative mRNA expression of NOS2, IL1B, and IL6 in THP-1 macrophages; H: Representative flow cytometry histograms of the CD80 expression levels in THP-1 macrophages; I: Quantification of FITC-CD80 mean fluorescence intensity; J-L: Representative immunoblots and the relative protein expression of AHR and CYP1A1 in THP-1 macrophages; M-O: Relative mRNA expression of AHR, CYP1A1, CYP1B1, and IL22 in THP-1 macrophages. The data are shown as the mean ± SD (n = 3 cell per group). Group comparisons were analyzed using one-way ANOVA with Sidak’s multiple comparisons test. ^aP < 0.01, ^bP < 0.001, ^cP < 0.0001. AHR: Aryl hydrocarbon receptor; NC: Negative control; LPS: Lipopolysaccharide; IDA: Trans-3-indoleacrylic acid; MFI: Mean fluorescence intensity.

Consistent with our in vivo findings, the results of the ELISA and RT-qPCR analyses revealed that the IDA intervention significantly suppressed the LPS/IFN-γ-induced upregulation of iNOS, IL-1β, and IL-6 expression. However, these anti-inflammatory effects were markedly attenuated following AHR knockdown (Figure 6B-G and Supplementary Figure 3B-G).

To further evaluate the regulatory effects of IDA on macrophage inflammatory responses in vitro, flow cytometry was performed to characterize macrophage polarization. CD80 and CD86 co-expression were used as indicators of M1 macrophages, whereas CD206 was used as a marker of M2 macrophages. IDA treatment significantly reduced the proportion of CD80⁺CD86⁺ M1 macrophages, while AHR knockdown largely abolished this inhibitory effect (Supplementary Figure 4A-D). Furthermore, analysis of the mean fluorescence intensity (MFI) of polarization-associated markers revealed that AHR knockdown attenuated the IDA-mediated reduction in CD80 expression, further supporting the involvement of AHR signaling in the inhibition of M1 macrophage activation (Figure 6H and I and Supplementary Figure 3H and I). In contrast, no significant differences were observed in either the number of CD206⁺ macrophages or CD206 MFI among the experimental groups, although a slight increase was observed following IDA treatment (Supplementary Figure 4E-H). These findings indicate that IDA suppresses proinflammatory M1 macrophage polarization through an AHR-dependent mechanism, while exerting limited effects on M2 macrophage differentiation.

We elucidated the molecular mechanism by which IDA inhibits M1 macrophage polarization by determining the expression levels of AHR and its downstream targets. IDA treatment significantly upregulated the protein levels of AHR and CYP1A1, as well as the mRNA expression levels of CYP1A1, CYP1B1, and IL22. These effects were markedly diminished following AHR knockdown (Figure 6J-O and Supplementary Figure 3J-O). Collectively, these findings indicate that IDA exerts macrophage-intrinsic anti-inflammatory effects through activation of the AHR signaling pathway.

IDA attenuates the DSS-induced disruption of the gut microbiota

16S rRNA gene sequencing was performed to investigate the effect of IDA on the gut microbiota composition. Alpha diversity analysis showed that, compared with DSS treatment, IDA treatment significantly increased microbial richness and diversity, as indicated by higher Chao1 and Shannon indices, as well as an increased number of observed OTUs (Figure 7A-C). PCoA revealed distinct clustering between the two groups, indicating that IDA administration restructured the gut microbial community (Figure 7D). Additionally, the Venn diagram shows 672 and 797 OTUs in the DSS and IDA groups, respectively, with 352 shared OTUs (Figure 7E). These results demonstrate that IDA increases the species richness and diversity of the gut microbiota in mice with colitis.

Figure 7 Trans-3-indoleacrylic acid attenuates the dextran sulfate sodium-induced disruption of the gut microbiota. A-C: Alpha diversity indices of the gut microbiota, including Chao1, Shannon, and observed operational taxonomic units (OTUs); D: Principal coordinate analysis plot based on Bray-Curtis distance; E: Venn diagram illustrating the shared and unique OTUs; F and K: Taxonomic composition of the gut microbiota at the phylum and genus levels; G-I: Relative abundance of specific phyla: Firmicutes, Proteobacteria, and Actinobacteriota; J: Ratio of relative abundance of Firmicutes to Bacteroidota; L-P: Relative abundance of specific genera: Akkermansia, Bifidobacterium, Lactobacillus, Escherichia-Shigella, and Parasutterella; Q: Linear discriminant analysis (LDA) effect size analysis identifying biomarker taxa (LDA score > 4.0). The data are shown as the mean ± SD (n = 6 mice per group). Group comparisons were analyzed using Mann-Whitney test. In D, the P value was determined by PERMANOVA. ^aP < 0.01. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; F/B: Firmicutes/Bacteroidota; N.D.: Not detected; LDA: Linear discriminant analysis; PCoA: Principal coordinate analysis.

Next, the taxonomic composition was analyzed. At the phylum level, Firmicutes and Bacteroidota were the predominant phyla (Figure 7F). Compared with the DSS group, the IDA group presented increases in the abundance of Firmicutes (14.74%) and Actinobacteriota (1.35%) and a decrease in the abundance of Proteobacteria (10.91%) (Figure 7G-I). Although the proportion of Bacteroidota remained relatively stable, IDA treatment increased the Firmicutes/Bacteroidota ratio (Figure 7J), a parameter that is typically reduced in mice with DSS-induced colitis. At the genus level, norank_Muribaculaceae and Odoribacter were relatively abundant in both experimental groups (Figure 7K). Notably, the abundances of Akkermansia and beneficial probiotics, including Bifidobacterium and Lactobacillus, were significantly increased in the IDA group (Figure 7L-N). Furthermore, the relative abundances of the potential pathobionts Escherichia-Shigella and Parasutterella were significantly lower in the IDA group than in the DSS group (Figure 7O-P).

We performed LDA effect size to identify the differentially abundant taxa between the groups. The IDA group exhibited the significant enrichment of multiple taxa within the phylum Firmicutes, including the class Clostridia, the family Ruminococcaceae, and the genus Lachnospiraceae_UCG-006 (LDA score > 4.0; Figure 7Q). Notably, these enriched taxa include bacterial groups associated with SCFA production and are commonly considered beneficial commensals. These findings suggest that IDA treatment is associated with an enrichment of bacteria with potentially beneficial metabolic characteristics.

IDA modulates SCFA metabolism and microbiota-SCFA interactions in mice with colitis

SCFAs are critical for intestinal homeostasis, and our 16S rRNA sequencing data revealed the enrichment of SCFA-producing taxa in the IDA group. SCFA concentrations in the cecal contents were quantified using gas chromatography-mass spectrometry to investigate whether IDA regulates intestinal SCFA metabolism. Compared with the DSS group, the IDA treatment group presented significantly elevated concentrations of total SCFAs, acetic acid (AA), and butyric acid (BA; Figure 8A-C). An increasing trend was also observed for the propionic acid (PA) level, although this difference did not reach statistical significance (Figure 8D).

Figure 8 Trans-3-indoleacrylic acid modulates short-chain fatty acid metabolism and microbiota-short-chain fatty acid interactions in mice with colitis. A-D: Concentrations of total short-chain fatty acids (SCFAs), acetic acid, butyric acid, and propionic acid in cecal contents; E: Spearman correlation heatmap between the differentially abundant bacterial genera and SCFAs; F: Correlation network analysis among colitis features, microbiota, and SCFAs. Node sizes represent the node degree. The data in A-D are shown as the mean ± SD (n = 5 mice per group). Five randomly selected mice from each group were included in the SCFA analysis due to insufficient fecal sample availability. Group comparisons were analyzed using Mann-Whitney test. In E and F, red indicates positive correlations, while blue indicates negative correlations. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. DSS: Dextran sulfate sodium; IDA: Trans-3-indoleacrylic acid; SCFAs: Short-chain fatty acids; PA: Propionic acid; VA: Valeric acid; AA: Acetic acid; BA: Butyric acid; IVA: Isovaleric acid; CA: Caproic acid; 2-BA: 2-methylbutyric acid; IBA: Isobutyric acid; DAI: Disease activity index; Abs(r): Absolute value of the correlation coefficient.

Spearman’s correlation analysis was subsequently performed to identify the microbial genera associated with these metabolic alterations. Among the top 20 SCFA-associated genera, beneficial commensals such as Lachnospiraceae_UCG-006, Akkermansia, and Lachnoclostridium exhibited strong positive correlations with AA, BA, PA, and valeric acid (VA) levels (Figure 8E). Conversely, the abundances of Escherichia-Shigella and Faecalibaculum were negatively correlated with the levels of these metabolites.

A relevance network was constructed to further characterize the associations among colitis features, SCFAs, and the gut microbiota. AA and BA presented the highest node degrees, suggesting that they occupy central positions within the association network constructed in the present study (Figure 8F). Specifically, the levels of AA and BA were significantly positively correlated with the abundances of microbial genera such as Lachnoclostridium, Quinella, and norank_Lachnospiraceae, along with changes in the colon length and body weight. In contrast, the levels of AA and BA were negatively correlated with the abundances of Escherichia-Shigella and Faecalibaculum, as well as the DAI score, spleen index, and the levels of inflammatory mediators. Furthermore, the abundances of these two proinflammatory taxa were negatively correlated with PA and VA levels but significantly positively correlated with the expression of Nos2 and Il6.

Collectively, these findings show that IDA treatment is associated with altered intestinal SCFA profiles and distinct microbiota-SCFA associations in mice with colitis.

DISCUSSION

UC is a chronic, idiopathic inflammatory disease of the intestinal mucosa, with an incidence rate that continues to increase annually[27]. Notably, persistent mucosal inflammation significantly predisposes patients to an increased risk of colorectal cancer (CRC)[28]. Currently, the management of UC primarily relies on 5-aminosalicylic acid, corticosteroids, and biological agents[29]. However, these therapies are often limited by adverse effects and high nonresponse rates, highlighting the need for more effective and sustainable treatment options. Recent research suggests that investigating gut microbiota-derived metabolites and their roles in UC pathogenesis may provide valuable insights into the development of therapeutic approaches[30,31]. In the present study, we demonstrated that IDA, a gut microbiota-derived indole metabolite, significantly attenuated DSS-induced acute colitis in mice. Concurrently, the IDA intervention ameliorated intestinal barrier dysfunction and increased intestinal mucin secretion. Mechanistically, IDA effectively suppressed intestinal M1 macrophage polarization and reduced the secretion of proinflammatory cytokines via activation of the AHR-CYP1A1 signaling axis. In addition, IDA regulated the gut microbiota composition and SCFA metabolism, as evidenced by an increased abundance of beneficial commensal bacteria and elevated levels of BA and AA.

The intestinal microenvironment in individuals with UC is characterized by dysbiosis of the gut microbiota and immune dysregulation. In this context, the macrophage population polarizes toward a proinflammatory phenotype, acting as a primary source of inflammatory cytokines and chemokines that perpetually exacerbate the mucosal injury[32,33]. Therefore, the targeted modulation of macrophages has emerged as a crucial intervention for improving the intestinal immune microenvironment. As a major class of tryptophan metabolites, indole and its derivatives have been shown to ameliorate immune dysregulation by modulating macrophage phenotypic switching. Yang et al[34] reported that IPA regulates macrophage polarization via the TGF-β1/Smad signaling pathway, which improves epithelial integrity in mice with colitis. Liu et al[35] reported that by inhibiting the TLR4/MyD88/NF-κB pathway in M1 macrophages, IAA restores the M1/M2 polarization balance and ultimately reduces aortic inflammation. In the present study, we found that IDA selectively attenuated proinflammatory M1 macrophage polarization without promoting M2 macrophage differentiation, suggesting that IDA may exert immunomodulatory effects primarily by restraining inflammatory macrophage activation rather than inducing an anti-inflammatory macrophage phenotype. These results were validated in both the DSS-induced colitis model in vivo and the LPS/IFN-γ-induced inflammation model in vitro. Notably, suppression of M1 polarization and enhancement of M2 polarization are not necessarily reciprocal or obligatorily coupled processes. In experimental colitis, adipose-derived stem cells reduced total and M1 macrophages while leaving the M2 macrophage population unchanged, whereas the flavonoid compound LZ205 suppressed M1-associated inflammatory responses without significantly affecting multiple M2-associated genes[36,37]. Myeloid-specific deletion of ornithine decarboxylase likewise altered M1-associated responses without corresponding changes in M2 markers or M2 macrophage abundance[38]. Conversely, COMP-angiopoietin-1 reduced overall macrophage infiltration together with both M1- and M2-associated indices, indicating that changes in macrophage subsets may also reflect regulation of total macrophage recruitment rather than a reciprocal phenotypic conversion[39]. Collectively, these findings indicate that macrophage responses following intervention may exhibit distinct patterns and do not invariably represent a reciprocal shift between M1 and M2 polarization states.

AHR is a ligand-activated transcription factor that mediates the regulatory effects of indole derivatives on various biological processes[40]. Under basal conditions, AHR remains in the cytoplasm in association with a multiprotein chaperone complex containing a heat shock protein 90 dimer, the cochaperone p23, AHR-interacting protein, and c-SRC protein kinase. Upon activation by ligands, AHR is translocated into the nucleus and forms a heterodimer with AHR nuclear translocator, thereby inducing the expression of target genes such as CYP1A1, CYP1B1, and IL-22[41]. AHR is widely expressed in barrier tissues and immune cells, where it contributes to the maintenance of intestinal homeostasis and the modulation of immune responses[42,43]. Indole derivatives activate AHR to promote macrophage polarization toward an anti-inflammatory phenotype and suppress proinflammatory responses, thus alleviating various inflammatory diseases, including periodontitis and systemic lupus erythematosus[44,45]. In the context of colitis, research on the interaction between indole and AHR has focused largely on intestinal barrier function. IAA, ILA, and IAld have been reported to reduce mucosal permeability and maintain epithelial integrity in experimental colitis[18,46,47]. Similarly, IDA exerts diverse protective effects on intestinal diseases; in addition to restoring intestinal barrier function, it also inhibits intestinal epithelial necroptosis to alleviate NEC[19,48]. However, little is known about its immunomodulatory role in the intestinal microenvironment, particularly with respect to macrophage polarization. In the present study, IDA restored the expression of AHR and its downstream targets, including CYP1A1 and CYP1B1, in proinflammatory macrophages. Since CYP1A1 and CYP1B1 are classical transcriptional targets of AHR, their upregulation further supports the activation of the AHR signaling pathway. Although CYP1A1 is widely regarded as a canonical marker of AHR activation, emerging evidence suggests that it may also participate in the regulation of intestinal inflammation. For example, CYP1A1 has been reported to be required for the protective effects of urolithin A on experimental colitis, as Cyp1a1-deficient mice failed to maintain intestinal barrier integrity and were not protected from intestinal inflammation[49]. In addition, CYP1A1 can metabolize endogenous substrates, such as estradiol, to generate 2-hydroxyestradiol, a metabolite with reported anti-inflammatory and antioxidant properties[50]. Nevertheless, whether these metabolic activities contribute to macrophage polarization or intestinal inflammation remains unclear, and the downstream signaling events linking CYP1A1 to the regulation of inflammation have yet to be elucidated. We further verified the involvement of AHR signaling by employing complementary pharmacological and genetic approaches in vivo and in vitro, respectively. In the DSS-induced colitis model, the administration of the AHR antagonist CH-223191 markedly attenuated the protective effects of IDA. In parallel, AHR knockdown in both THP-1 macrophages and BMDMs consistently abolished the inhibitory effects of IDA on M1 macrophage polarization and inflammatory mediator production. Collectively, these findings provide complementary pharmacological and genetic evidence that IDA suppresses proinflammatory macrophage responses predominantly through AHR signaling.

Compared with that in healthy individuals, the gut microbiota composition in patients with UC is significantly altered and is characterized by a decreased abundance of bacteria that are beneficial for intestinal homeostasis, the overgrowth of potential pathogens, and a reduction in microbial diversity[51]. Indole derivatives have been shown to reshape the gut microbiota in patients with UC[52,53]. Our results revealed marked differences in the microbial community composition between the DSS and IDA groups. IDA treatment significantly increased the richness and diversity of the gut microbiota in mice with colitis, suggesting that IDA was associated with an improvement in UC-associated microbial dysbiosis. Moreover, the IDA group was characterized by an enrichment of various bacteria associated with SCFA metabolism, such as Akkermansia, Bifidobacterium, Lactobacillus, and Lachnospiraceae_UCG-006. Bifidobacterium and Lactobacillus are widely recognized as therapeutic probiotics. They ferment dietary fibers to produce lactate, which is rapidly metabolized by butyrate-producing bacteria into butyrate or propionate[54-56]. Moreover, Lactobacillus participates in tryptophan metabolism and is capable of generating various indole derivatives, suggesting a possible reciprocal relationship between Lactobacillus enrichment and indole derivative production following IDA treatment[57]. Akkermansia exploits intestinal mucins as a nutrient source, degrading them into utilizable monosaccharides that are further fermented to produce acetate and propionate. This process also stimulates goblet cell mucus secretion, thus promoting mucus turnover and improving intestinal barrier integrity[58]. In UC patients, particularly those in the active phase, the abundance of Akkermansia is markedly decreased, which may be attributable to the disruption of the intestinal mucus layer[59]. Furthermore, the IDA intervention significantly reduced the relative abundance of proinflammatory pathobionts, such as Escherichia-Shigella and Parasutterella. These genera are markedly enriched in patients with UC and are implicated in promoting the progression of both UC and colitis-associated CRC[60,61]. Multiple in vivo studies have shown that the composition of the gut microbiota influences both mucosal susceptibility to inflammation and the subsequent progression of colitis[62-64]. Accordingly, strategies designed to restore a beneficial gut microbiota, including fecal microbiota transplantation (FMT) and supplementation with beneficial microbial metabolites, may represent promising therapeutic approaches for alleviating colitis.

In addition to tryptophan metabolites, SCFAs represent another class of metabolites with significant protective effects on the host. SCFAs provide essential energy for colonocytes and enhance intestinal barrier function by alleviating mucosal inflammation and promoting the expression of TJ proteins[65,66]. Our 16S rRNA sequencing results showed that IDA increased the abundance of several bacterial taxa known to produce SCFAs, suggesting that IDA may influence intestinal SCFA metabolism. The quantification of SCFAs revealed that the IDA intervention significantly increased the concentrations of AA, BA, and PA. Among these SCFAs, BA has attracted considerable attention because of its beneficial role in maintaining intestinal homeostasis. In colonic epithelial cells, butyrate serves as a primary energy source via β-oxidation, thus sustaining an anaerobic luminal environment. Such an anaerobic state favors the growth of obligate anaerobes, such as butyrate-producing bacteria, while limiting the expansion of oxygen-tolerant bacteria such as E. coli, ultimately fostering a healthy gut microbial ecosystem[67,68]. In addition, a study demonstrated that BA could directly bind to AHR in the human intestinal epithelial cell lines HT-29 and Caco-2 and upregulate the expression of AHR, CYP1A1, and AHR repressor[69]. These findings underscore the protective role of BA in maintaining intestinal homeostasis. A subsequent correlation analysis suggested that AA and BA might serve as central metabolites in the IDA-regulated gut microbial metabolic network. The concentrations of AA, BA, PA, and VA were significantly positively correlated with the abundances of Lachnoclostridium, Quinella, and norank_Lachnospiraceae. Notably, these genera are recognized as SCFA-producing taxa and are considered beneficial members of the gut microbiota[70-73]. A recent study revealed that Dendrobium huoshanense polysaccharides could alleviate UC by promoting SCFA metabolism in Lachnoclostridium edouardi, highlighting the therapeutic potential of Lachnoclostridium in treating colitis[74]. However, research concerning the specific roles of Quinella and norank_Lachnospiraceae in colitis remains limited and requires further investigation.

In summary, this study revealed the protective effect of IDA on UC, and its detailed mechanism was closely associated with the regulation of macrophage polarization and the activation of the AHR-CYP1A1 signaling pathway. From the perspective of the 3Rs principle, our experimental design incorporated appropriate group allocation and multiple outcome assessments to maximize the information obtained from each animal. However, several limitations should be acknowledged. In our work, colitis was induced via the administration of DSS in the drinking water, a model that primarily disrupts the intestinal epithelial barrier to trigger innate immunity, thus mimicking acute mucosal injury. Conversely, the TNBS model relies on hapten-mediated activation to induce a Th1/Th17-driven adaptive immune response, effectively recapitulating immune-mediated transmural inflammation. Since the DSS and TNBS models reflect distinct aspects of UC pathogenesis, subsequent studies using a TNBS-induced colitis model are needed to comprehensively evaluate the protective effects of IDA across different immunopathological contexts. In addition, the present study used an acute colitis model, and the long-term efficacy and safety of IDA were not evaluated. Future studies using chronic colitis models together with comprehensive preclinical toxicological assessments will further support the potential clinical translation of IDA. Furthermore, this study primarily used pharmacological inhibition to assess the ability of AHR to mediate the IDA-induced suppression of M1 macrophage polarization. However, its administration in vivo may introduce off-target effects or influence other cell types. Therefore, the generation of macrophage-specific AHR knockout mice is imperative for future research to genetically confirm whether IDA-mediated macrophage regulation relies on AHR signaling. Moreover, although CYP1A1 was examined as a classical downstream target reflecting AHR activation, the precise mechanisms by which CYP1A1 may participate in the regulation of inflammatory responses remain unclear. Further studies are needed to elucidate the downstream signaling events linking CYP1A1 to macrophage polarization and intestinal inflammation. Finally, although our results indicate that IDA reshapes the gut microbiota composition and alters fecal SCFA profiles, these findings represent associations rather than direct evidence of causality. Future studies employing approaches such as FMT or germ-free mouse models will help clarify whether microbiota-derived changes contribute to the protective effects of IDA on colitis.

CONCLUSION

In summary, our study demonstrates that IDA alleviates colitis by selectively suppressing proinflammatory M1 macrophage polarization through AHR signaling, while being associated with favorable alterations in gut microbiota composition and SCFA profiles (Figure 9). These findings highlight IDA as a potential microbiota-derived therapeutic candidate for UC.

Figure 9 Proposed mechanism of trans-3-indoleacrylic acid-mediated protection against dextran sulfate sodium-induced colitis. Trans-3-indoleacrylic acid activates aryl hydrocarbon receptor signaling to suppress pro-inflammatory M1 macrophage polarization, thereby alleviating intestinal inflammation and is associated with favorable alterations in gut microbiota composition and short-chain fatty acid profiles. IDA: Trans-3-indoleacrylic acid; DSS: Dextran sulfate sodium; AHR: Aryl hydrocarbon receptor; ARNT: Aryl hydrocarbon receptor nuclear translocator; SCFA: Short-chain fatty acid; AIP: Aryl hydrocarbon receptor-interacting protein.

ACKNOWLEDGEMENTS

The authors would like to thank the Xiangya School of Public Health, Central South University, for providing the experimental instruments and facilities for this study.