Microbiota-derived IPA alleviates intestinal mucosal inflammation through upregulating Th1/Th17 cell apoptosis in inflammatory bowel disease

Abstract

The gut microbiota-derived metabolite indole-3-propionic acid (IPA) plays an important role in maintaining intestinal mucosal homeostasis, while the molecular mechanisms underlying IPA regulation on mucosal CD4⁺ T cell functions in inflammatory bowel disease (IBD) remain elusive. Here we investigated the roles of IPA in modulating mucosal CD4⁺ T cells and its therapeutic potential in treatment of human IBD. Leveraging metabolomics and microbial community analyses, we observed that the levels of IPA-producing microbiota (e.g. Peptostreptococcus, Clostridium, and Fournierella) and IPA were decreased, while the IPA-consuming microbiota (e.g. Parabacteroides, Erysipelatoclostridium, and Lachnoclostridium) were increased in the feces of IBD patients than those in healthy donors. Dextran sulfate sodium (DSS)-induced acute colitis and CD45RB^highCD4⁺ T cell transfer-induced chronic colitis models were then established in mice and treated orally with IPA to study its role in intestinal mucosal inflammation in vivo. We found that oral administration of IPA attenuated mucosal inflammation in both acute and chronic colitis models in mice, as characterized by increased body weight, and reduced levels of pro-inflammatory cytokines (e.g. TNF-α, IFN-γ, and IL-17A) and histological scores in the colon. We further utilized RNA sequencing, molecular docking simulations, and surface plasmon resonance analyses and identified that IPA exerts its biological effects by interacting with heat shock protein 70 (HSP70), leading to inducing Th1/Th17 cell apoptosis. Consistently, ectopic expression of HSP70 in CD4⁺ T cells conferred resistance to IPA-induced Th1/Th17 cell apoptosis. Therefore, these findings identify a previously unrecognized pathway by which IPA modulates intestinal inflammation and provide a promising avenue for the treatment of IBD.

Keywords: HSP70; Indole-3-propionic acid; Th1 cells; Th17 cells; mucosal inflammation.

Graphical abstract

Figure 1.

Signature alterations in tryptophan-metabolizing microbiota and metabolites in the feces of IBD patients. (a), (b) KEGG pathway enrichment analysis of differentially accumulated metabolites. (a) Pathway enrichment in CD vs. healthy individuals. (b) Pathway enrichment in UC vs. healthy individuals. (c) Comparison of tryptophan metabolite levels in the feces of IBD patients and healthy individuals. (d) Relative content of IPA in IBD patients and healthy individuals. (e) PCoA plots of the bacterial communities (16S rRNA gene amplicons) in IBD patients and healthy individuals. (f) Relative abundance of IPA-producing and IPA-consuming bacteria in the feces of IBD patients and healthy individuals. (g) Fold changes in IPA-producing microbiota (blue) and IPA-consuming microbiota (red) in the feces of IBD patients compared with healthy controls. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Oral administration of IPA alleviates DSS-induced acute colitis in mice. DSS-induced acute colitis was performed in WT (n = 10) mice. Mice were given IPA (50 mg/kg, prepared in 1 M NaOH, final pH 7.0) by oral gavage daily from 0 to 10 days of DSS treatment. An equivalent volume of H₂O vehicle was served as control. All mice were sacrificed on day 10. (a) Changes in body weight over a 10-day experimental period were indicated as a percentage of the original weight at the start of the experiment. (b) Changes in the disease activity index (DAI) in four groups during an experimental period were recorded and shown in the chart. (c) Gross morphology of the large bowels on day 10 after DSS exposure. (d) Colon length was measured and recorded. The pathological scores of colonic sections were shown in the chart. (e) Histological appearance of colonic sections after H&E staining. Scale bars: 100 μm. (f) LPMCs were separated from WT mice receiving DSS treatment, and flow cytometric analysis was performed to determine the intracellular expression of TNF-α, IFN-γ, IL-10, and IL-17A. (g) Percentages of TNF-α⁺CD4⁺ T cells, IFN-γ⁺CD4⁺ T cells, and IL-17A⁺CD4⁺ T cells are shown in the bar chart. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Oral administration of IPA effectively ameliorates chronic colitis in mice induced by the adoptive transfer of CD45RB^highCD4⁺ T cells. Splenic CD45RB^highCD4⁺ T cells were isolated from WT mice by utilizing flow cytometry and cell sorting and injected intraperitoneally into 8-week-old Rag1^–/– mice (5 × 10⁵ cells/mouse, n = 10/group). Mice received IPA (50 mg/kg, prepared in 1 M NaOH, final pH 7.0) via oral gavage every other day throughout the entire experimental process. An equivalent volume of H₂O vehicle was served as control. (a) and (b) Mice were weighed weekly and DAI was calculated after T cell transfer. (c) and (d) Mice were sacrificed at week 8 after T cell transfer, and colon morphology and length were shown. (e) Pathological scores of the colonic tissues were calculated, and representative colon sections were stained with H&E. Scale bars: 100 μm. (f) LPMCs were harvested from the colons of Rag1^–/– mice transferred with CD45RB^highCD4⁺ T cells, and intracellular expression of IL-17A, IFN-γ, and TNF-α in LP-CD4⁺ T cells was analyzed by flow cytometry. (g) Percentages and numbers of IL-17A⁺CD4⁺, IFN-γ⁺CD4⁺, and TNF-α⁺CD4⁺ T cells were shown in the chart. Statistical analyses were performed with unpaired Student’s t-tests. The data were presented as mean ± SEM. The data were representative of three independent experiments. Statistical analysis was performed using Tukey’s multiple comparison test (a), (b), and (d) and the unpaired Student’s t-test. (d) and (g). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

IPA compromises Th1 and Th17 cell differentiation in IBD. Peripheral blood CD4⁺ T cells isolated from healthy individuals (n = 11), CD patients (n = 12), and UC patients (n = 15) were stimulated in vitro with IPA (100 μM) for 2 or 5 days. The expression of inflammatory cytokines in CD4⁺ T cells was analyzed via qRT-PCR and flow cytometry analysis, respectively. (a-c) Expression of inflammatory cytokines in peripheral blood CD4⁺ T cells from healthy individuals, CD patients, and UC patients. (d) Peripheral blood CD4⁺ T cells from healthy individuals (n = 6) were isolated and cultured under different polarizing conditions. The expression of inflammatory cytokines was assessed. (e) Statistical analysis of the terminally differentiated CD4⁺ T cells in healthy individuals. (f) The expression of inflammatory cytokines in differentiated cells was measured by qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

IPA decreases HSP70 expression in CD4⁺ T cells from IBD patients. CD4⁺ T cells were isolated from peripheral blood of IBD patients and healthy individuals (n = 3/group) and sequenced following IPA treatment. (a) The heat map illustrates the differentially expressed genes between IPA-treated and untreated groups. (b) Target genes with a Spearman correlation coefficient greater than 0.8 in RNA-seq data and the IPA potential binding protein database. (c) The expression of HSPA1A mRNA in peripheral blood CD4⁺ T cells from IBD patients and healthy individuals (n = 8/group) was detected by qRT-PCR. (d, e) Surface plasmon resonance analysis revealed that IPA binds to HSP70 protein. (f) T cells isolated from peripheral blood of five healthy individuals were cultured under Th0-, Th1-, Th2-, Th17- and Treg-polarizing conditions, respectively, for 5 days, and HSP70 expression was assessed by flow cytometry assay. (g) CD4⁺ T cells isolated from peripheral blood of five healthy individuals were cultured under Th0-, Th1-, Th2-, Th17- and Treg-polarizing conditions, respectively, for 5 days, and HSPA1A mRNA expression was quantified using qRT-PCR. (h) Immunohistochemistry demonstrated HSP70 staining in the colonic mucosa of healthy individuals and active IBD patients (original magnification: ✕200), and quantified the number of HSP70⁺ cells per HP field. The data were represented as the mean ± SEM. Statistical analyses were performed using Spearman correlation analysis (b), unpaired Student’s t-test, two-way ANOVA, Tukey’s multiple comparison test (c), and ordinary one-way ANOVA (g and h). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

IPA disrupts mitochondrial integrity and promotes apoptosis in CD4⁺ T cells. (a-c) CD4⁺ T cells from peripheral blood of IBD patients and healthy individuals were stimulated in vitro with IPA (100 μM) for 5 days. Cells were collected and stained with Mito-Tracker Green, Mito-Tracker Deep Red (a), or JC-1 (b) for 30 minutes. The stained cells were then analyzed by flow cytometry, and the results were statistically summarized in (c). (d) Representative scanning electron microscopy images of CD4⁺ T cells and their mitochondria after IPA stimulation. The images were taken at magnifications of × 3.0k (upper panel) and × 8.0k (lower panel). (c) Based on the data from (d), a quantitative analysis was performed to determine the ratio of viable mitochondria to apoptotic mitochondria per field of view. (e) The dynamic changes in glycolysis of IPA-treated CD4⁺ T cells from peripheral blood of IBD patients and healthy individuals were measured using a Seahorse extracellular flux analyzer. (f) The dynamic changes in mitochondrial stress of IPA-treated CD4⁺ T cells from peripheral blood of IBD patients and healthy individuals were also assessed using a Seahorse extracellular flux analyzer. (g) Western blot analysis of CD45RB^highCD4⁺ T cells treated with or without IPA in vivo showed the expression levels of P-Ask1, Ask1, Hsp70, P-Jnk, Jnk, Pro-Caspase 9, Cleaved Caspase 9, Pro-Caspase 3, and Cleaved Caspase 3 caspase, respectively. Statistical analyses, including unpaired Student’s t-test, two-way ANOVA, and ordinary one-way ANOVA, were performed to validate these observations. The data were presented as the mean ± SEM, with significance indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7.

Overexpression of HSPA1A promotes Th1/Th17 cell immune response in IBD patients and healthy individuals. (a, b) Peripheral blood CD4⁺ T cells (1 × 10⁵ cells/well) were isolated from 8 healthy individuals, 8 active Crohn’s disease patients, and 8 active ulcerative colitis patients. The cells were then transduced with lentiviruses expressing shRNA targeting HSPA1A (LV-shHSPA1A), HSPA1A (LV-HSPA1A), or a negative control (LV-NC). These cells were co-cultured with plate-bound anti-CD3 mAb (5 μg/mL) and soluble anti-CD28 mAb (2 μg/mL) for 5 days. The frequencies of IL-17A- and IFN-γ-expressing CD4⁺ T cells were analyzed by flow cytometry. (c) The RNA expression levels of various genes in the transduced CD4⁺ T cells were measured using qRT-PCR. The levels of inflammatory cytokines secreted by the transduced CD4⁺ T cells were detected using ELISA. The data were presented as the mean ± SEM. Statistical analyses were evaluated using Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.