Akkermansia muciniphila Protects Against Trinitrobenzene Sulfonic Acid Induced Colitis by Inhibiting IL6/STAT3 Pathway

Abstract

Background

Inflammatory bowel disease is a long-standing inflammatory disorder that influences the intestinal tract. The intent of this research is to explore whether the relative abundance of Akkermansia muciniphila is related to the IL6/STAT3 pathway and the fundamental molecular mechanisms of A. muciniphila on a trinitrobenzene sulfonic acid (TNBS)-induced enteritis mouse model, including the expression of inflammatory cytokines and proteins in the IL6/STAT3 signaling pathway.

Methods

The association between the A. muciniphila and IL6/STAT3 was investigated by using mucosal biopsies and fecal samples. TNBS-induced colitis mouse models were performed to elucidate the underlying mechanisms. The alteration of intestinal microbiota was organized by 16s rRNA sequencing.

Results

In Crohn’s disease patients, the level of STAT3 and IL-6 presented a negative relationship with A. muciniphila. The expression of IL-6, p-STAT3, and STAT3 was downregulated in A.m+TNBS group, indicating A. muciniphila may inhibit the IL6/STAT3 pathway in TNBS-induced enteritis in vivo. To investigate the potential defensive role of A. muciniphila supplementation in vivo with TNBS-induced enteritis, 16S rRNA sequencing was performed to analyze changes in the intestinal microbiota composition. The results revealed a marked increase in microbial diversity and abundance within the A. muciniphila-treated group, suggesting a beneficial modulation of the gut microbiome associated with the supplementation.

Conclusions

Our findings declared that A. muciniphila supplementation alleviates gastrointestinal inflammation through IL-6/STAT3 signaling pathway. This protective effect was mediated by the downregulation of the IL-6 and STAT3, highlighting a potential mechanism by which A. muciniphila modulates inflammatory responses. This work disclosed that A. muciniphila demonstrates a defensive influence against TNBS-induced enteritis in vivo, proposing it qualified as a unique therapeutic focusing on modulating IL-6, STAT3, or p-STAT3 in the treatment of colitis.

Graphical Abstract

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Introduction

Ulcerative colitis (UC) and Crohn’s disease (CD) belong to inflammatory bowel disease (IBD), which is a long-term gastrointestinal mucosa inflammatory disorder that can affect the whole intestinal tract.¹ This disorder can present with classic signs of bellyache, bloody diarrhea, and extraintestinal manifestations. However, the exact causes and underlying mechanisms of this disease remain poorly understood. Several speculations suggest that the interaction between genetic risk factors, intestinal microbiota dysbiosis, environment, and immune system dysregulation causes the development of IBD.² An environmental exposure can trigger gut microbiota dysbiosis in an individual with high genotype factors, prompting a dysregulated immune response and leading to progressive intestinal inflammatory alterations.

The intestinal microbiota plays an essential role in forming an inflammatory microenvironment, which, in return, influences and alters the composition of the microbial community. This bidirectional interaction highlights the complex association between inflammation and microbial dynamics in the gastrointestinal tract. Akkermansia muciniphila is a prominent and highly abundant bacterial species within the human bacteria, known for its noteworthy position in maintaining gut homeostasis and its specific association with the mucosal layer of the gastrointestinal tract, which has been identified as one of the promising next-generation probiotics (NGPs).³ Decreased level of A. muciniphila is associated with various inflammatory diseases, such as IBD, metabolic disorders (obesity and diabetes), and neurological disorders (multiple sclerosis, epilepsy, and melancholia).^4–6 Alteration of intestinal flora could induce primary resistance to immune checkpoint inhibitors. According to metagenomics of patient stool samples, the high abundance of A. muciniphila is related to good clinical responses to ICIs. Akkermansia muciniphila supplementation reinstated the efficiency of PD-1/PD-L1 obstruct in a manner reliant on interleukin-12, enhancing the infiltration of CCR9⁺CXCR3⁺CD4⁺ T lymphocytes into the neoplasm microenvironment in mice.⁷ Besides, previous data revealed that oral supplementation with A. muciniphila reduces obesity, glucose tolerance, hypercholesterolemia, and alcoholic or immune-related liver diseases in mice.^8,9 In summary, these findings suggest that A. muciniphila has beneficial immunomodulatory and anti-inflammatory properties in the gastrointestinal tract.

A practical molecule from the Janus kinase-STAT pathway, signal transducer and activator of transcription 3 (STAT3), is broadly hyperactivated in numerous cell types through a tyrosine phosphorylation signaling mechanism. The activation of STAT3 is often connected to the progress of immunodeficiency and autoimmune disorders, highlighting its importance in regulating immune responses and inflammation.^10,11 STAT3 performs a pivotal function in controlling immune responses by blocking the immune stimulation molecules while enhancing the production of factors that suppress immune responses. This dual action is necessary for sustaining an equilibrium between pro-inflammatory and anti-inflammatory responses.¹² Interleukin 6 (IL-6) can be combined with its receptor (IL-6R), which then contacts with the glycoprotein 130 (gp130) receptor, directly activating the JAK family of tyrosine kinases, which phosphorylate and activate STAT3 on the cell surface. Notably, the IL6/STAT3 pathway can also be activated as a result of gut dysbiosis. A recent study established that gut dysbiosis which led to increased gut permeability and elevated levels of intertumoral lipopolysaccharides could promote prostate malignancy advance through the stimulation of the IL-6/STAT3 signaling pathway in vivo.¹³ Therefore, inhibition of the IL6/STAT3 pathway may be achieved by improving gut dysbiosis.

Earlier research works have revealed that the onset of IBD is influenced by a combination of factors, including gut microbiota dysbiosis, alterations in intestinal immune responses, and the excessive release of inflammatory cytokines. These interconnected processes disrupt the delicate balance of the gastrointestinal environment, contributing to chronic inflammation and disease progression.¹⁴ There is growing evidence highlighting the essential position of the IL6/STAT3 signaling pathway in regulating inflammation, particularly in the context of IBD.^15,16 The IL6/STAT3 pathway promotes the survival and proliferation of intestinal epithelial cells (IECs), elevates levels of IL-6 can lead to persistent inflammation, tissue damage, and dysregulation of epithelial cell function.¹⁷ Given its crucial role in IBD, targeting the IL6/STAT3 pathway, blocking IL-6 or inhibiting STAT3 activation has been explored as a potential therapeutic strategy for treating IBD. However, there are currently no approved medications specifically targeting the IL-6/STAT3 pathway for the treatment of IBD, the pathway remains an area of significant interest in therapeutic research. To date, only a limited number of STAT3 inhibitors have entered clinical or preclinical development, primarily focusing on various cancers, including breast cancer, liver cancer, and lung cancer. Despite these hurdles, ongoing research into STAT3 inhibitors for cancer may pave the way for future exploration into IBD and other inflammatory diseases.

Taken together, A. muciniphila, as one of the most promising NGPs, is a therapeutic agent that alters intestinal microbiota and has an impact on inflammatory disorder development. The IL6/STAT3 pathway plays a critical position in maintaining the integrity and functionality of the intestinal barrier. The purpose of this study is to investigate the potential association between A. muciniphila and IL-6/STAT3. It aims to elucidate the underlying molecular mechanisms by which A. muciniphila affects a trinitrobenzene sulfonic acid (TNBS)-induced enteritis in vivo, focusing on its impact on the expression of key inflammatory molecules within the IL-6/STAT3 pathway. In addition, the alterations of gut microbiota would also be detected to explore the gut homeostasis balance effect of A. muciniphila. We aimed to verify A. muciniphila as a potential therapeutic strategy in IBD.

Materials and Methods

Subjects

Mucosal biopsies and fresh fecal samples were gathered from healthy participants (n = 12) and CD patients (n = 12) at the Department of Gastroenterology of the West China Hospital between April 2021 and December 2022. The identification of CD was established following the ECCO-ESGAR Guideline, which incorporates a comprehensive assessment of clinical history, physical examination, endoscopy findings, small bowel imaging, radiological evaluations (CT, MRI, or ultrasound), blood test results, and histological evidence.¹⁸ Baseline demographic information and disease characteristics of the healthy participants and CD patients were obtained from the medical records (Table 1). This study including human volunteers was evaluated and approved by the ethical committee of West China Hospital (Approval Number: 2024-501).

Experimental Mouse Models

Male C57BL/6 mice, aged 8-10 weeks, weighing 23-28 g, were obtained from Beijing Huafukang Bioscience Corporation (Beijing, China). IL-6^−/− mice, were purchased from Shanghai Model Organisms Center. All mice were hosted in specific pathogen-free conditions at West China Hospital BSL-2 animal facility. All mice were randomized into experimental groups and acclimatized for at least 7 days before the beginning of the experiment. Acute colitis was induced via a TNBS enema (2.5%, w/v) in vivo after 21 days of administrating with A. muciniphila or phosphate-buffered saline (PBS). The mice were indiscriminately allocated to 5 groups: the C57BL/6 control group (CTRL, n = 6), the C57BL/6 mice treated with PBS group (Ctrl + TNBS, n = 6), the C57BL/6 A.muciniphila-treated group (A.m + TNBS, n = 6), the IL-6^-/- mice treated with PBS group (IL-6^−/− + PBS, n = 4) and the IL-6^−/− mice treated with A. muciniphila group (IL-6^−/− + A.m, n = 4). Mice in the A.m + TNBS/IL-6^−/− + A.m and Ctrl + TNBS/ IL-6^−/− + PBS groups were administered A. muciniphila (3 × 10⁹ CFU of A. muciniphila in 200 μL of PBS) and PBS (200 μL) via gavage 14 days before TNBS treatment, respectively. All the groups were administered a TNBS enema on day 8. All the experimental mice were sacrificed on day 12. Colon pieces were quickly frozen in liquid nitrogen for 24 hours and subsequently stored at −80°C for further analysis. All the animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan, China (Approval Number: 20230226057).

Assessment of Disease Activity Index Score

The Disease Activity Index (DAI) score, a quantitative measure that includes parameters such as body weight loss, stool consistency, and the presence of hematochezia, was recorded daily to evaluate the severity of colitis-related damage.¹⁹ The following parameters were recorded daily during the experimental period: (1) body weight loss (0, no loss; 1, 1%-5% loss; 2, 6%-10% loss; 3, 10%-18% loss; or 4, >18% loss), (2) stool consistency (0, normal; 1, soft but still formed; 2, soft; 3, very soft, wet; or 4, watery diarrhea), and (3) blood (0, negative hemocult; 1, negative hemoccult; 2, positive hemocult; 3, blood traces in stool visible and 4, gross rectal bleeding).

Assessment of Histological (HAI) Score

The rectum (approximately 0.6 ± 0.2 cm) tissues were fixed in 10% paraformaldehyde at room temperature for at least 24 hours. Sections were paraffin-embedded, sectioned, and stained with hematoxylin–eosin staining for pathological analysis, conventionally. The light microscope was used to assess histological changes within histology scores (Histology Activity Index [HAI]), including inflammation damage, infiltrating mononuclear cells, and loss of goblet cells.¹⁹

ELISA Methods

Serum was achieved from the experimental mice, and the concentrations of IL-6, as well as the proteins STAT3 and p-STAT3, were quantified using an ELISA kit (ThermoFisher Scientific).

Western Blot Analysis

Colon tissues were collected, and proteins were obtained by using RIPA lysis buffer (Solarbio). The concentration of the target proteins was measured with a bicinchoninic acid (BCA) protein assay kit. A total of 20 µg of target protein was applied to a 10% SDS-PAGE gel for separation, followed by transfer onto PVDF membranes (Millipore) for subsequent analysis. The specific membranes were blocked with QuickBlock PBST (Beyotime) for 10 minutes, followed by overnight incubation with primary antibodies against STAT3, and p-STAT3 at 4 °C. Afterward, the membranes were gestated with specific secondary antibodies for another 1 hour. Targeting protein bands were detected by manipulating Immobilon Western HRP substrate (Merck KGaA) and analyzed by ChemiDocTM MP imaging system (Bio-Rad).

RNA Preparation and Real-Time Quantitative PCR Analysis

Total RNA was obtained from the rectal tissue by consuming the Tissue Total RNA Isolation Kit (Vazyme) according to the manufacturer’s directions. Reverse transcription was accomplished by consuming HiScript III RT SuperMix for qPCR (Vazyme). Quantitative real-time PCR was managed by consuming the QuantStudio 7 Pro Real-Time PCR System (ThermoFisher Scientific) to analyze gene expression.

Immunofluorescence

STAT3, p-STAT3, IL-6, and epithelial cadherin (E-cad) expression in colonic biopsy specimens were evaluated via immunofluorescence. Paraffin specimens were minced into 5-μm-thick fragments and adhered to adhesion microscope slides. After pretreatment, sections were incubated with STAT3 (Cell Signaling Technology), p-STAT3 (Cell Signaling Technology), IL-6 (Proteintech), and E-cad (Proteintech) antibodies at 4 °C nightlong. Segments were then incubated with the specific Alexa 488-conjugated (STAT3 1:200, p-STAT3 1:200, and IL-6 1:400)/550-conjugated (E-cad 1:400)/550-conjugated (E-cad) secondary antibodies (Cell Signaling Technology) for 45 minutes at room temperature. 4',6-Diamidino-2-Phenylindole (DAPI), which stained the nucleus needs to incubate at room temperature for 5 minutes. Imageries were photographed using a fluorescence microscope (Nikon) or the VS200 whole slide imaging system (Olympus) and investigated by ImageJ software.

Akkermansia muciniphila Cultivation

Akkermansia muciniphila (ATCC BAA-835T) was cultured by inoculating 1 mL of bacterial suspension into 300 mL of culture medium containing 4 g BHI, 0.03 g L-cysteine, and 0.15 g mucin in 100 mL ddH₂O. The culture was incubated under anaerobic conditions for 48 hours in an anaerobic workstation. After incubation, the bacteria were harvested by high-speed centrifugation, washed twice with PBS, and the optical density at 600 nm (OD600) was measured. The bacterial suspension was adjusted to 3.0 × 10⁹ CFU/mL in PBS and stored at −80 °C for further use.

Cell Culture

Human IECs (HIEC-6, ATCC, CRL-3266) were cultured in DMEM medium supplemented with 10% fetal bovine serum (SenBeiJia Biological Technology), 1% penicillin/streptomycin (Sigma-Aldrich). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. For experimental treatment, cells were divided into 2 groups. Ctrl group: cells were treated with 1 μg/mL lipopolysaccharide (LPS) for 6 hours to induce an inflammatory response. Ctrl + A.m group: cells were pretreated with both 1 × 10⁸ CFU/mL A. muciniphila and 1 μg/mL LPS for 6 hours. After treatment, cells were collected for further analysis.

DNA Extraction From Mouse Colon Contents

Colon contents were obtained from individual mice and groups after euthanasia and kept at −80 °C until further administering. The preparation and sequencing of the specific colon sections were carried out following the instructions of the AllPrep DNA/RNA Mini Kit (Qiagen). The extracted bacterial DNA was quantified using quantitative real-time PCR and then collected at −80 °C for the following analyses.

16S rRNA Sequencing

The resulting DNA was sequenced and selected for microbial diversity detection, focusing on the V3–V4 regions of the 16S rRNA gene. The microbiota 16S rRNA V3–V4 amplification primers used were 515F and 806R. The PCR reaction was performed using Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs). Sequencing was accomplished on the Illumina NovaSeq platform, generating 250 bp paired-end reads for further analysis.

Statistical Analysis

All data were expressed means ± standard deviation. Statistical analyses of mouse groups were conducted by consuming unpaired Student’s t-test, Mann–Whitney U test, or one-way analysis of variance (ANOVA), as appropriate. Spearman correlation analysis was employed to assess relationships between variables. Statistical significance was indicated by asterisks: *P < .05, **P < .01, ***P < .001. GraphPad Prism v.7.04 and SPSS version 20.0 for Windows were used for statistical analysis.

For microbiota analysis, paired-end reads were merged using FLASH (VI.2.7,http://ccb.jhu.edu/software/FLASH/) to get raw tags.²⁰ Raw tags were processed, checked, and filtered using Qiime (v1.9.1, http://qiime.org/scripts/split_libraries_fastq.html) to generate clean tags.^21,22 These tags were then compared with the species annotation database (Silva database, https://www.arb-silva.de/) to detect and remove chimeric sequences, ensuring the final valid data (effective tags). The average sequencing depth after quality control was 67 183 reads (with a minimum of 60 456 reads and a maximum of 74 719 reads). Raw reads were further filtered and clustered into Operational Taxonomic Units (OTUs), followed by de novo OTU picking at 97% pair-wise identity using the UCHIME algorithm (http://www.drive5.com/uparse/).^23,24

Results

A. muciniphila Presented Negatively Associated With STAT3 and IL-6 in CD Patients

To reveal the clinical significance of A. muciniphila and CD, we assessed the abundance of A. muciniphila in CD (n = 12) and healthy control (HC, n = 12; Table 1, Figure 1A). Among the CD patients, 6 were under mild disease (MCD), another 6 were under severe disease (SCD). Next, we measured the relative abundance of A. muciniphila in the fresh fecal samples collected from both HC and CD patients. The results showed that A. muciniphila was downregulated in CD patients compared to HC(Figure 1B). We also detected the expression of IL6/STAT3 pathway in the endoscopic intestinal tissue samples from the HC and CD patients and observed a decrease in the levels of IL-6 and STAT3 expression in CD patients (Figure 1C and D). However, the levels of IL-6 and STAT3 displayed no significant difference between mild and severe CD patients (Figure 1E and F). Correlation analysis was managed to investigate the connection between IL6/STAT3 pathway and A. muciniphila. Interestingly, the result suggested an inverse connection between A. muciniphila and the expression of IL-6 and STAT3 (Figure 1G and H). Specifically, higher levels of A. muciniphila were connected with lower levels of IL-6 and STAT3. Consequently, our results indicate that A. muciniphila colonization was decreased in CD patients, and its abundance was negatively connected with IL-6 and STAT3.

To investigate the changes and analyze the localization of the IL6/STAT3 pathway expression during CD, immunofluorescence was implemented on paraffin sections of colonic biopsies from the CD patients with high or low A. muciniphila abundance. Dual immunofluorescence staining was utilized to co-localize the IEC marker epithelial cadherin (E-cad) with IL6/STAT3 pathway markers IL-6, p-STAT3, and STAT3 in their intestinal tissues. According to the immunofluorescence analysis, CD patients with low A. muciniphila abundance exhibited significantly higher levels of IL6/STAT3 pathway compared to those with high A. muciniphila abundance in IECs (Figure 1I–L). This result suggested that A. muciniphila may inhibit the IL6/STAT3 pathway in IECs.

Oral Forced Feeding With A. muciniphila Presented a Defensive Influence Against TNBS-Induced Colitis

To explore whether A. muciniphila contributed to the abrogation of intestinal inflammation, we used the TNBS-induced intestinal inflammation in vivo. C57BL/6J mice in the A.m + TNBS and Ctrl + TNBS groups were managed with heat-killed A. muciniphila or PBS, respectively (Figure 2A). In colitis models, the disease severity is characteristically correlated with weight loss, HAI, and DAI scores.¹⁹ The percentage of weight change was deliberate daily over a 13-day period, with the baseline body weight recorded on day 0 for comparison. The A.m + TNBS group was suggestively protected from TNBS-induced colitis, showing reduced body weight loss (P < .05) and an increased DAI score (P < .05) (Figure 2B and C). Consistent with body weight loss and colon length shortening were also indirectly related to disease severity. Competed to the Ctrl + TNBS group, the A.m + TNBS group presented a longer colon (P < .05, Figure 2D and E). Histopathological examination of the rectal section revealed more severe epithelial erosion, a greater loss of goblet cells, and an increased number of infiltrating mucosal and submucosal leukocytes in the Ctrl + TNBS group compared to the A.m + TNBS group (Figure 2F), resulting in higher histological activity index scores (P < .001, Figure 2G) for mucosal inflammation. These results indicate that oral forced feeding with A. muciniphila provides protective effects against TNBS-induced colitis and alleviates colonic injury.

A. muciniphila Inhibited the IL6/STAT3 Pathway in TNBS-Induced Enteritis

A previous study highlighted the significance of STAT3 and p-STAT3 in shaping and regulating innate immunity, revealing its deletion during hematopoiesis leads to myeloid cell irregularities and triggers CD-like pathogenesis.¹⁰ To investigate the probable mechanism essential to the protecting effects of A. muciniphila, we then evaluated IL6/STAT3 pathway activity in colon tissues. Subsequent analyses confirmed that A. muciniphila supplementation downregulated the IL6/STAT3 pathway at both the protein and mRNA levels, including reduced levels of IL-6, p-STAT3, and STAT3. Western blot analysis confirmed that both STAT3 and p-STAT3 in the A.m + TNBS group were suggestively decreased compared to those in the Ctrl + TNBS group (Figure 3A and B). To further validate this conclusion, we examined STAT3 and p-STAT3 using ELISA, RT-qPCR, and immunofluorescence in mice treated with or without A. muciniphila following TNBS-induced colitis. The expression levels of the IL-6 were evaluated by ELISA and RT-qPCR. As shown in Figure 3C–E, A. muciniphila supplementation inhibited IL6/STAT3 pathway in the protein level. The immunofluorescence intensity of STAT3 and p-STAT3 showed the same results (Figure 3H and I). RT-qPCR analysis confirmed that A. muciniphila supplementation could decrease IL-6 expression in mRNA level (Figure 3F). However, no significant changes had been observed in STAT3 (Figure 3G). To further validate these findings, we performed in vitro experiments using HIEC-6 cells. The results demonstrated that A. muciniphila decreased IL-6/STAT3 expression in both mRNA and protein level (Figure S1). Collectively, these findings confirm that A. muciniphila could inhibit the IL6/STAT3 pathway in TNBS-induced enteritis in vivo.

To further elucidate the mechanism by which A. muciniphila inhibits the IL-6/STAT3 signaling pathway in the treatment of enteritis, we utilized IL-6^−/− mice under TNBS-induced colitis conditions. The method used to establish the enteritis model was identical to that used for C57BL/6 mice, as illustrated in Figure 2A. Our findings revealed that IL-6^−/− mice exhibited a similar severity of intestinal inflammation regardless of the presence or absence of A. muciniphila supplementation, including body weight change, colon length, DAI, and HAI scores (Figure 4A–F). Subsequently, we evaluated IL6/STAT3 pathway activity in these colon tissues. Compared to IL-6^−/− + PBS group, no significant differences were observed in the inhibition of the IL-6/STAT3 pathway. The levels of STAT3 remained unchanged between the two groups, whether assessed at the RNA or protein level (Figure 4G–N). These data demonstrated the inhibitory effect of A. muciniphila on the IL-6/STAT3 pathway, highlighting a novel and effective approach to alleviating intestinal inflammation, and positioning it as a valuable addition to the treatment strategies for IBD.

A. muciniphila Improved Gut Dybiosis in TNBS-Induced Enteritis

The gut microbiota is a key element in the controlling of intestinal inflammation. To further investigate the protective effect of oral forced feeding with A. muciniphila in TNBS-induced enteritis, 16S rRNA sequencing was performed to evaluate changes in the intestinal microbiota composition. This analysis aimed to identify any microbial shifts associated with A. muciniphila treatment and their potential link to the alleviation of colitis symptoms. We performed 16s rRNA profiling of the rectum from A.m + TNBS (n = 8) and Ctrl + TNBS groups (n = 9). At the both phylum and species levels, the average composition of microbes in the A.m + TNBS group was more abundant to Ctrl + TNBS group (Figure 5A and B). The A.m + TNBS group disclosed a greater relative abundance of various phyla, such as Actinobacteria, Verrucomicrobiota, Bacteroidota, and Campylobacter. Consistently, most Ctrl + TNBS samples clustered together, whereas A.m + TNBS samples were mixed based on the phylum composition (Figure 5C and D). To explore the differentiations in species complexity and structural changes in bacteria communities, both alpha diversity and unweighted UniFrac beta-diversity metrics were used to assess the overall microbial structure. The results revealed significant differences between the A. muciniphila (A.m + TNBS) and control (Ctrl + TNBS) groups. The richness of the bacteriome in rectal samples from A. muciniphila-treated mice was significantly higher compared to the control group (Figure 5E, P = .0192). Principal coordinate analysis (PCoA) of the microbiome structure showed varying degrees of overlap between the cohort clusters (Figure 5F). Overall, these data indicate that A. muciniphila supplementation enhances both α-diversity and β-diversity, significantly influencing the abundance of specific bacterial populations in the gastrointestinal tract.

The gut microbiota of the A.m + TNBS and Ctrl + TNBS groups were distinctly divided into 2 bunches, indicating that continuous A. muciniphila supplementation improved gut dysbiosis. To further examine the metagenomic functions associated with microbial abundance differences between these 2 groups, we conducted Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Administration of A. muciniphila may alter cellular processes, metabolism, genetic information processing, and organismal systems. In addition, a lack of A. muciniphila may cause human disease (Figure 5G–J). These findings highlight the essential role of A. muciniphila in gut dysbiosis.

Discussion

Dysbiosis of the gut microbiota is one of the most critical elements in the development of IBD.^25,26 Although the specific mechanisms remain unknown, it is generally believed that lower diversity and abundance of the intestinal microbiota are related to IBD.^27–29 Several clinical trials have investigated the therapeutic efficacies of prebiotics in IBD. Prebiotics, such as Bifidobacterium, Lactobacillus, Clostridium butyricumand, and Streptococcus thermophilus, have been used as supplements to support the growth of commensal bacteria and sustain IBD treatment.³⁰ A randomized, double-blinded, double-dummy trial recruited 327 patients from 60 hospitals in 10 European countries to evaluate the efficacy of the probiotic preparation Escherichia coli Nissle 1917 compared to traditional mesalazine treatment in maintaining remission. This study verified that E. coli Nissle 1917 presented greater relapse-preventing effectiveness (40/100, 36.4%) than mesalazine (38/112, 33.9%) over 12 months in patients with UC in remission. Also, there was no difference in safety between the 2 groups.³¹ Another multicenter, randomized, double-blinded, placebo-controlled trial evaluated the ability to induce remission of a high-potency probiotic, VSL#3 (Lactobacilli, Bifidobacteria, and S. thermophilus) in 147 UC patients. Mild-to-moderately active patients with UC were separated into 2 groups (VSL#3 and placebo control), compared with the placebo-control group, VSL#3 group presented with a significantly higher improvement in UCDAI score at both week 6 (25/77, 32.5%) and week 12 (33/77, 42.9%). Hence, supplementation with prebiotics is a safe and effective therapeutic strategy to succeed a clinical response and remission in patients with IBD.

Akkermansia muciniphila is an obligate anaerobic bacterium known for its ability to degrade mucin, a glycoprotein component of mucus. This species was initially identified and isolated in 2004 from the fecal samples of healthy adult individuals. Thereafter, 16S rRNA sequencing revealed that this bacterium belongs to the phylum Verrucomicrobia with 92% sequence similarity. Akkermansia muciniphila is the sole characteristic member of the Verrucomicrobia phylum discovered in the human gastrointestinal tract, where it constitutes approximately 3%-5% of the intestinal microbiota under normal conditions.³² Latest research works have exhibited that lack or decrease of this NGPs was associated with various disorders, especially colitis and colorectal tumors. However, the specific mechanisms remain inconclusive. As in colitis, the increased presence of A. muciniphila in the gastrointestinal tract resulted in an enhanced expression of the intestinal transcription factor cAMP-responsive element-binding protein H (CREBH). This transcription factor, in coordination with microRNAs miR-143 and miR-145, facilitated the renaissance of IECs and accelerated the repair of wounds in the gastrointestinal lining. This regenerative process was mediated through the activation of insulin-like growth factor (IGF) and insulin-like growth factor-binding protein 5 (IGFBP5) signaling pathways, which are critical for maintaining intestinal health and recovery. Moreover, Amuc_1100 also had an effect on activating CREBH, inhibiting intestinal endoplasmic reticulum pressure and enriching the manifestation of genes contained in intestinal barrier reliability and IEC reinforcement.³³ Consistence with this observation, previous studies also revealed Amuc_1100 can decrease the presence of sensitive macrophages and CD8+ T cells in the intestinal environment. Additionally, it can also engage with Toll-like receptor 2 (TLR2), contributing to the strengthening of the gut barrier function.³⁴  Akkermansia muciniphila induced the production of mucus and enhanced the expression of Reg3γ in the colon, which contributed to the remodeling of the microbiota. It led to a reduction in serum endotoxin levels and a decrease in the expression of Toll-like receptors in the islets. Additionally, it stimulated an increase in Treg cells within the islets and elevated the levels of IL-10 and TNF-β in the pancreatic lymph nodes.³⁵ Threonyl-tRNA synthetase (AmTARS), one of the aminoacyl-tRNA synthetases that secreted by A. muciniphila, perform a crucial position in modulating immune reactions by stimulating the polarization of macrophages toward the M2 phenotype. This polarization enhances the production of IL-10. The process is driven by the activation of the MAPK and PI3K/AKT signaling pathways, which converge on the transcription factor CREB, ultimately amplifying IL-10 production. Concurrently, this mechanism suppressed NF-κB, a central mediator of inflammation, reducing inflammatory responses and promoting immune homeostasis.³⁶ As in colorectal tumors, Amuc_2172 performs a significant position in controlling colorectal malignancy cell function. This protein gained access to colorectal cells through micropinocytosis by acting as an acetyltransferase and targeting Lys14 on histone H3 (H3K14ac). The increased levels of H3K14 acetylation, especially at the Hspa1a gene loci, facilitated the transcription and subsequent exudation of heat-shock protein 70 (HSP70) in intestinal tumor cells. The increased HSP70 levels enhanced the immune stimulation of cytotoxic T lymphocytes (CTLs) not only in vitro but also in vivo, contributing to a more effective antitumor immune response. Additionally, bioengineered nanoparticles have been developed to safely and efficiently deliver Amuc_2172, demonstrating promise as a drug delivery system for malignance management in a homograft mice model. It highlighted the therapeutic potential of Amuc_2172 in enhancing immune responses and targeting colorectal tumors.³⁷  Akkermansia muciniphila can also be used in other cancer immunotherapy. According to Luo’s research, A. muciniphila-derived extracellular vesicles enlarged the amount of granzyme B-positive CD8+ and IFN-γ+CD8+ T cells and M1-like macrophages in vitro, indicating that A. muciniphila-derived extracellular vesicle is a promising immunotherapeutic strategy for prostate cancer.³⁸ Outer membrane vesicles (OMVs), released from A. muciniphila, performed several vital functions to support gastroenterology health. First, maintaining intestinal microbiota homeostasis: OMVs selectively promoted the growth of beneficial gut bacteria through a mechanism involving membrane fusion, assisting to re-establish a healthy microbial community and restoring microbiota balance. Second, enhancing immune response: OMVs were translocated into Peyer’s patches to stimulate the activation of B cells and dendritic cells, leading to a robust mucosal immunoglobulin A (IgA) response, which is crucial for neutralizing pathogens and maintaining mucosal immunity. Third, reserving intestinal barrier integrity: OMVs entered IECs and boosted the illustration of tight junction proteins and mucus production, strengthening the gut barrier, preventing pathogen invasion and ensuring gut homeostasis.³⁹ Hence, A. muciniphila has been supposed a hopeful probiotic for the management of numerous diseases.

Various research implies that STAT3/IL-6 signaling performs an essential position in the improvement and pathogenesis of IBD. p-STAT3 enables its translocation to the nucleus which enriches STAT3 initiation, thereby driving Th17 cell differentiation. In mouse models, both pharmacological inhibition of APT2 and genetic knockout of Zdhhc7 (encoding DHHC7, facilitates STAT3 membrane recruitment and phosphorylation) effectively alleviated colitis symptoms, highlighting potential therapeutic methods.⁴⁰ After, those outcomes revealed that aiming the STAT3/IL-6 signaling pathway may be an operative therapy in patients with IBD.

To clarify whether and how A. muciniphila has the potential therapeutic role in CD. We detected the expression of IL6/STAT3 signaling pathway in biopsy samples and the relative abundance of A. muciniphila in fecal samples from CD patients. Our results indicated that A. muciniphila colonization is negatively associated with IL6/STAT3 patyway, suggesting A. muciniphila may inhibit the IL6/STAT3 in IECs. Then we used C57 mice in the presence of TNBS solution to construct acute enteritis models. In the acute colitis model, we administered A. muciniphila orally to mice, followed by TNBS dissolution liquid-induced enteritis model. Our results showed that body weight loss, HAI,and DAI score were expressively advanced in the control group. Moreover, we verified whether A. muciniphila regulates the STAT3/IL-6 pathway in mice. Our result exhibited that expressing of IL-6, p-STAT3, and STAT3 was downregulated in A.m + TNBS group, indicating A. muciniphila may inhibit the IL6/STAT3 pathway in TNBS-induced colitis. These results indicated that oral gavage with A. muciniphila could protect against TNBS-induced colitis and alleviate colonic injury through IL6/STAT3 pathway.

The gut dysbiosis is a significant element in gastrointestinal mucous inflammation and immune homeostasis. 16S rRNA sequencing was conducted to further explore the alterations influence of A. muciniphila supplementation in TNBS-induced enteritis. The abundance of the microbiota was notably increased in mice that were administered A. muciniphila. Compared to the Ctrl + TNBS group, the abundance of Actinobacteria, Verrucomicrobiota, Bacteroidota, and Campylobacter increased, and that of Fimicutes and Paraperlucidibaca decreased after oral administration of A. muciniphila. Additionally, the microbiota profiles of the intestinal mucosa in both the A.m + TNBS group and Ctrl + TNBS group were noticeably separated into 2 distinct bunches. This separation suggested that continuous A. muciniphila supplementation effectively improved gut dysbiosis. Previous research works have also informed an increase in the abundance of a strong ability to mucus-degrading bacteria and a decrease in the abundance of less mucus-degrading bacteria, such as A. muciniphila, in IBD patients.⁴¹ A few researchers have suggested that A. muciniphila may stimulate goblet cell to produce more mucin by degrading mucin in the intestine. On the one hand, it can reduce excessive mucin deposition in the host body. On the other hand, the newly produced mucin provides a more favorable environment and nutrition for the survival of A. muciniphila.⁴² The 2 reinforce each other, creating a positive feedback effect. According to Lu’s metabolomics analysis,⁴³ a total of 30 key metabolites were identified as highly predictive for differentiating between the A. muciniphila and control groups. Among them, tryptophan (Trp) levels were significantly decreased in the A. muciniphila group, indicating its potential role in metabolic regulation. Principal coordinate analysis (PLS-DA) further confirmed that the gut metabolite composition was significantly altered after A. muciniphila intervention. Overall, A. muciniphila has not been studied at the genomic level, and the interaction between specific mechanism, gut microbiota composition, and disease progression of intestinal inflammation needs to be investigated further using advanced techniques.

In conclusion, we demonstrated that supplementation with A. muciniphila may help improve mucosal inflammation through microbe–host interactions that downregulate the IL6/STAT3 signaling pathway. This study reveals that A. muciniphila offers a defensive influence against TNBS-induced enteritis in vivo, suggesting it is a potential therapeutic target for modulating IL6/STAT3 signaling in the treatment of colitis.

Acknowledgments

Beijing Novogene Bioinformatics Technology Co., Ltd. provided sequencing and data analysis services.

Author Contributions

M.J.: Writing—original draft preparation, review and editing; Z.Z., C.M., Y.J., Y.W., H.G.: Writing—review and editing; H.Z.: Supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Sichuan Province Science and Technology Support Program [2023YFS0279, 2024YFFK0347].

Conflicts of Interest

All of the authors claimed no potential conflict of interest

Ethical Considerations

The collection of colonic samples and the analysis of clinical data were approved by the ethics committee of West China Hospital (Approval Number: 2024-501). All animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan, China (Approval Number: 20230226057).

Data Availability

Data are available on reasonable request. The microbiota data sets have been deposited in NCBI Sequencing Read Archive (Accession ID: PRJNA983528).

References