High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

Abstract

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a frequently encountered disorder characterized by voiding symptoms and pelvic or perineal pain. Proinflammatory T helper 17 (Th17) cells are essential for triggering the development of CP/CPPS. High-salt diet (HSD) consumption has been found to cause an accumulation of sodium chloride in peripheral organs, inducing autoimmune responses via the Th17 cell axis. It is currently unknown whether HSD affects the etiology and course of CP/CPPS.

Methods: Patients diagnosed with CP/CPPS were evaluated with the National Institutes of Health Chronic Prostatitis Symptom Index scoring system, and the correlation between the symptoms of CP/CPPS with HSD was analyzed. The experimental autoimmune prostatitis (EAP) mouse was established and the mice were fed either a normal-salt diet (NSD) or HSD for 6 weeks to investigate the impact of HSD on CP/CPPS. Then, 16S ribosomal RNA sequencing and untargeted metabolomics were introduced to detect the differences in the gut microflora composition and metabolite profiles between NSD-fed and HSD-fed mice, followed by fecal microbiota transplantation, 5-hydroxyindole acetic acid (5-HIAA) supplementation, aryl hydrocarbon receptor (AHR) inhibition, and in vitro Th17 differentiation experiments, which were performed to explore the mechanisms underlying HSD-aggravated CP/CPPS. Finally, chromatin immunoprecipitation assay and quantitative polymerase chain reaction were conducted to validate whether AHR can serve as a transcription factor by interacting with the serum and glucocorticoid-regulated kinase 1 (Sgk1) promoter in CD4⁺ T cells.

Results: Increased salt consumption had a positive correlation with symptom scores of CP/CPPS patients, which was validated by feeding EAP mice with HSD, and HSD worsened the prostate inflammation and tactile allodynia in EAP mice through promoting the differentiation of CD4⁺ T cells to Th17 cells. HSD exacerbated EAP by significantly reducing the relative abundance of beneficial gut microflora, such as Lactobacillaceae, and gut microbiota metabolite 5-HIAA, which is related to tryptophan metabolism. The prostate inflammation, tactile allodynia, and proportion of Th17 cells in mice that received fecal suspensions from the EAP + HSD group were significantly more severe or higher than those in mice that received fecal suspensions from the EAP + NSD group. However, 5-HIAA supplementation ameliorated the symptoms of EAP caused by HSD through inhibiting the differentiation of CD4⁺ T cells to Th17 cells, while AHR inhibition abrogated the protective effects of 5-HIAA supplementation on EAP mice fed a HSD through promoting the differentiation of CD4⁺ T cells to Th17 cells. Mechanistically, it has been revealed that the SGK1/forkhead box protein O1 (FOXO1) pathway was significantly activated during cytokine-induced Th17 cell differentiation, and AHR has been shown to inhibit SGK1 transcription by interacting with the Sgk1 promoter in CD4⁺ T cells to inhibit FOXO1 phosphorylation, consequently restoring the equilibrium of Th17 cell differentiation.

Conclusion: Our findings indicated that high salt intake represented a risk factor for the development of CP/CPPS as it promoted the differentiation of CD4⁺ T cells to Th17 cells through the 5-HIAA/AHR/SGK1/FOXO1 axis, which might be a potential therapeutic target for CP/CPPS.

Keywords: 5-Hydroxyindole acetic acid (5HIAA); Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS); High-salt diet; Serum and glucocorticoid-regulated kinase 1 (SGK1); Th17 cells.

Fig. 1

Severe CP/CPPS symptoms were positively correlated with HSD. a Heatmap illustrating the relationship between sodium consumption and clinical characteristics. b The sodium intake of CP/CPPS patients was greater than that of healthy controls. Sodium intake increased with NIH-CPSI scores, quality of life scores, urination score, pain or discomfort score, and age. ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001; ns non-significant. CP/CPPS chronic prostatitis/chronic pelvic pain syndrome, BMI body mass index, HSD high-salt diet

Fig. 2

HSD promoted Th17 cell development and worsened prostate inflammation in EAP mice. a Simple schematic diagram of the implementation plan of the experiment. b HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). c Tactile allodynia development in the 4 groups of mice (n = 4). d The levels of IL-1β, TNF-α, IL-17A, and IFN-γ in the serum of the mice in the 4 groups (n = 4). e Proportion of Th1 and Th17 cells of CD4⁺ T cells detected by flow cytometry in the splenic lymphocytes of vaccinated mice from those 4 groups (n = 4). f The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns non-significant. BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IFN-γ interferon-γ, IL-17A interleukin-17A, DAPI 4',6-diamidino-2-phenylindole, Th T helper

Fig. 3

Metabolites and microflora compositions were dissimilar between EAP mice fed an HSD and an NSD. a Bacterial 16S rRNA fecal sample α-diversity indexes for mice in the EAP + HSD and EAP + NSD groups. b Comparison of the gut microflora composition between mice in the EAP + HSD and EAP + NSD groups. c Heatmap of metabolites in serum of mice in the EAP + HSD and EAP + NSD groups. d Metabolites that were differentially abundant in the serum of mice in the EAP + HSD or EAP + NSD group. e Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enriched pathways associated with the differentially abundant metabolites of NOD mice between the EAP + HSD and EAP + NSD groups. The tryptophan metabolism abnormalities were indicated in red. f Integrated association analysis of the 16S rRNA and LC–MS data of the mice in the EAP + HSD and EAP + NSD groups. ^*P < 0.05, ^**P < 0.01; ns non-significant. NOD nonobese male diabetic/LtJ, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, LC–MS liquid chromatography-mass spectrometry, TRP tryptophan, FDR false discovery rate, CCA canonical correlation analysis

Fig. 4

Transplantation of fecal microbiota from EAP mice with HSD elicited more severe prostate inflammation in psuedogerm-free mice. a Simple schematic diagram of the experimental workflow. b HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). c Development of tactile allodynia in mice belonging to the 4 groups (n = 4). d The concentrations of IL-1β, TNF-α, IL-17A, and 5-HIAA in the serum of NOD mice in the 4 groups were measured (n = 4). e Proportion of Th17 of CD4⁺ T cells, as determined by flow cytometry, among the splenic lymphocytes of immunized mice in the 4 groups (n = 4). f The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, FMT fecal microbiota transplantation, HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, DAPI 4',6-diamidino-2-phenylindole

Fig. 5

5-HIAA supplementation ameliorated the symptoms of EAP caused by HSD. a The levels of 5-HIAA secreted into the serum of the mice were measured in 4 groups: Ctrl + NSD, Ctrl + HSD, EAP + NSD, and EAP + HSD groups (n = 4). b Simple schematic diagram of the experimental workflow. c HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). d Tactile allodynia development in NOD mice in the 4 groups (n = 4). e Secretion levels of IL-1β, TNF-α, and IL-17A in the serum of mice in the 5 groups (n = 4). f Flow cytometry was used to determine the proportion of Th17 of CD4⁺ T cells among the splenic lymphocytes of immunized mice in the 5 groups (n = 4). g The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, DAPI 4',6-diamidino-2-phenylindole

Fig. 6

Inhibition of AHR abrogated the protective effects of 5-HIAA supplementation on EAP mice fed an HSD. a AHR levels in prostate tissues of mice among the Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, and EAP + HSD + 5-HIAA groups (n = 4). b A simple schematic diagram of the experimental workflow. c HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). d Tactile allodynia development in the mice from the Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, EAP + HSD + 5-HIAA, and EAP + HSD + 5-HIAA + CH groups (n = 4). e The levels of IL-1β, TNF-α, and IL-17A in the serum of the mice in the 6 groups (n = 4). f Proportion of Th17 of CD4⁺ T cells, as determined by flow cytometry, among the splenic lymphocytes of immunized mice in the 6 groups (white arrowheads, n = 4). g The infiltration of Th17 cells in prostate tissues from mice was evaluated by immunofluorescence (scale bar = 100 μm, n = 4). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, CH CH223191 (an AHR inhibitor), HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, AHR aryl hydrocarbon receptor, DAPI 4',6-diamidino-2-phenylindole

Fig. 7

HSD promoted the differentiation of Th17 cells by activating SGK1/FOXO1 signaling pathway. a The in vitro Th17 cell differentiation ratio was determined by flow cytometry (n = 3). b Th17 cell differentiation ratio was determined in an in vitro differentiation experiment using immunofluorescence (scale bar = 100 μm, n = 3). c SGK1, p-FOXO1, and FOXO1 levels in prostate tissues from Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, and EAP + HSD + 5-HIAA mice (n = 4). d AHR, SGK1, p-FOXO1, and FOXO1 levels in prostate tissues of mice in the EAP + HSD + 5-HIAA and EAP + HSD + 5-HIAA + CH groups (n = 4). e AHR, SGK1, p-FOXO1, and FOXO1 levels in naïve CD4⁺ T cells of Ctrl, Sti, Sti + HSD, Sti + HSD + 5-HIAA, Sti + HSD + 5-HIAA + CH groups (n = 3). f Transcriptional levels of Sgk1 in naïve CD4⁺ T cells of Ctrl, Sti, Sti + HSD, Sti + HSD + 5-HIAA, Sti + HSD + 5-HIAA + CH groups (n = 3). g For the ChIP-qPCR assay, 3 potential enhancer segments of the mouse promoter region of Sgk1 were created. h qPCR was used to assess the immunoprecipitated DNA by a ChIP assay (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns non-significant. EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, AHR aryl hydrocarbon receptor, SGK1 serum and glucocorticoid-regulated kinase 1, FOXO1 forkhead box protein O1, CH CH223191 (an AHR inhibitor), ChIP chromatin immunoprecipitation, qPCR quantitative polymerase chain reaction, DAPI 4’,6-diamidino-2-phenylindole, Sti stimulation with IL-6, IL-23, TGF-β, anti-IFN-γ, and anti-IL-4

Fig. 8

Schematic diagram illustrating the function and mechanism of HSD in the progression of CP/CPPS. HSD elevated the levels of SGK1 by inhibiting the 5-HIAA/AHR axis and further induced the phosphorylation of FOXO1, consequently promoting RORγt-driven Th17 cell differentiation to exacerbate CP/CPPS. HSD high-salt diet, CP/CPPS chronic prostatitis/chronic pelvic pain syndrome, 5-HIAA 5-hydroxyindole acetic acid, AHR aryl hydrocarbon receptor, SGK1 serum and glucocorticoid-regulated kinase 1, FOXO1 forkhead box protein O1, RORγt retinoic acid receptor-related orphan receptor gamma-t