Akkermansia Muciniphila Ameliorates Imiquimod-Induced Skin Thickening, Colitis, and Gut Microbiota Alterations: A Metagenome Association Study

Abstract

A decreased abundance of fecal Akkermansia muciniphila (Akk) has been observed in patients with psoriasis and psoriatic arthritis. The potential beneficial effects of Akk in managing psoriasis have been proposed, yet results remain inconsistent and mechanisms unclear. Using imiquimod (IMQ)-treated C57BL/6 mice, we conducted a metagenomic association study of pasteurized Akk (pAkk) in the IMQ mice through whole-genome shotgun sequencing. We also performed a dextran sodium sulfate (DSS)-induced colitis experiment and an intestinal permeability test. The association among pAkk supplements, skin thickness, inflammatory profiles, fecal microbiota alterations, functional genetic predictions, intestinal epithelium inflammation, and barrier integrity was investigated. The study demonstrated that pAkk supplementation ameliorated IMQ-induced skin thickening, weight loss, spleen weight gain, serum IL-17A, TNF-α levels, and DSS-induced colitis. pAkk supplementation was linked to greater fecal microbial diversity and alterations in fecal microbiota composition, with increased prevalence of Muribaculaceae, Bifidobacterium pseudolongum, Desulfovirionaceae, Erysipelotrichaceae, and Alistipes ihumi, which have been implicated in the Gamma-Aminobutyric Acid (GABA) shunt, cholinergic synapse, cell cycle, and Mitogen-Activated Protein Kinase (MAPK) pathways. In conclusion, pAkk may mitigate IMQ-induced skin thickening and DSS-induced colitis, associated with reduced levels of TNF-α and IL-17A. pAkk supplementation alters fecal microbiota and metabolic pathways in IMQ-treated mice.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02436-9.

Introduction

Psoriasis is a Th17-medicated chronic inflammatory disease with a prevalence of up to 3% in the United States [1, 2]. Severe psoriasis is usually characterized by widespread skin lesions with thick scales and arthritis that may cause significant physical and psychological burdens. [1] Severe psoriasis has been associated with multiple comorbidities, including cardiovascular diseases, metabolic syndrome, depression, and inflammatory bowel diseases (IBD). [3] In addition to shared genetic background and common inflammatory pathways, gut microbiota alterations have been proposed to play a role in the pathogenesis of psoriasis and associated comorbidities. [4–7]

A decreased diversity of gut microbiota has been reported in patients with psoriasis and psoriatic arthritis [4, 5]. The distinct fecal microbiota of psoriasis has been associated with an increased fecal and serum inflammatory cytokines, [4] treatment response and disease severity [8]. Akkermansia muciniphila (Akk) has been proposed to be a gut microbiota signature in psoriasis and psoriatic arthritis [9]. As a Gram-negative anaerobic bacterium, Akk colonizes the mucosal layer of the intestinal epithelium. Intestinal Akk prevalence has been negatively associated with active IBD disease and positively associated with IBD remission [10, 11], suggesting a beneficial role of Akk in modulating intestinal inflammation. In addition to maintaining intestinal integrity, Akk has been involved in immune homeostasis, preventing insulin resistance, obesity,¹² periodontitis [13], and metabolic dysfunction [12], and enhancing the effect of anticancer therapies [14]. The effect of Akk in psoriatic diseases is worth further exploration.

This study investigated the effect of Akk on skin inflammation, colitis symptoms, intestinal epithelium inflammation, and gut microbiome profiles using the imiquimod (IMQ)-treated mice model. The IMQ–induced psoriasis-like mouse model is a fast, reliable method for studying psoriasis and testing therapies [15]. In brief, topical application of 5% IMQ cream (e.g., Aldara) to shaved skin or ears of susceptible strains (BALB/c or C57BL/6) for 5–7 days activates TLR7, driving IL-23/IL-17–mediated inflammation that mimics human plaque psoriasis with redness, scaling, epidermal thickening, and dermal immune cell infiltration. We next examined the association between Akk supplementation and alterations in circulating cytokines. We performed the pipelines of phylogenetic analysis, functional gene analysis, and pathway analysis to investigate the association among these profiles.

Materials and Methods

Animal Management

All experimental procedures were approved by the Institutional Animal Care and Utilization Committee (IACUC) of Taichung Veterans General Hospital (La-1091737) and performed in accordance with institutional guidelines. Male C57BL/6JNarl mice, aged four weeks, from the National Laboratory Animal Center (Taipei, Taiwan) were used for experiments. These mice were initially housed in separate cages (3–5 mice per cage) with free access to water and food. The mice were provided standard chow (5001, Laboratory Rodent Diet, USA). The housing conditions included a constant temperature of 22 ± 1 °C and a regular 12-h light/dark cycle (lights on 8 PM to 8 AM, with bedding Snai-chip #1511, Young Li). After 3 days of acclimatization and 2 days of antibiotics, the 5-week-old mice were fed standard chow with Akk supplements (5001, Laboratory Rodent Diet) or regular drinking water for 14 days. The 7-week-old mice were then reallocated to separate experiments.

Before the experiment, the mice were randomly distributed among the groups to ensure a comparable average body weight in each group. (Supplementary Fig. 1) Investigators were not blinded when performing the experiments or analyses.

IMQ Treatment and Administration

A daily topical application of 80 mg of commercially available IMQ cream (5%) (Aldara; 3 M Health Care, Leicestershire, UK) was applied to the shaved back and right ear for seven consecutive days, providing a daily dose of 4 mg of the active compound [15]. Dulbecco’s phosphate-buffered saline (PBS), concentration 1X (Gibco 14,190–144), was applied to the shaved back and right ear as a control.

Antibiotics

Since we do not have germ-free mice for study, we used an alternative protocol involving antibiotics. Briefly, to mitigate the background gut microbiota signals from experiments, Ciprofloxacin (0.2 g/L) (17,850, Sigma) + Metronidazole (1 g/L)(9,002,409, Cayman) [16] were added to drinking water two days before pasteurized Akk (pAkk) supplement or normal drinking water.

Pasteurized Akkermansia Muciniphila Administration

Both live Akk and pAkk were safe in mice, with the pasteurized form offering greater stability and more substantial immune and metabolic benefits, while the live form more effectively enhanced gut microbial homeostasis. [17, 18] Given its superior immune and metabolic benefits alongside stability as a non-viable form, so we chose pAkk for these experiments [17, 18]. Mice were orally gavaged with 100 µL of a suspension containing 1.25 × 10 [9] colony-forming units (CFUs)/mL pAkk supplements daily for 2 weeks prior to the experiment. pAkk BCRC 81048 was purchased from the Food Industry Research and Development Institute, Taiwan. Briefly, Akk BCRC 81048 was inactivated by pasteurization for 30 min at 70ºC water bath and was immediately frozen and stored at − 80° C [12]. The final product of pAkk BCRC 81048 was resuspended in phosphate-buffered saline with 2.5% glycerol at the specified concentration mentioned above, making it ready for subsequent use.

Fluorescein Isothiocyanate (FITC)-Dextran Intestinal Permeability Assay and Dextran Sodium Sulfate (DSS)-Colitis Induction

To investigate the effects of pAkk on intestinal permeability and DSS-induced colitis [19] in IMQ-treated mice, we first conducted a pilot study by dividing mice into eight groups (N = 5 in each group) (Fig. 1), to ensure the feasibility of combining the two experiments. After a 14-day pretreatment of oral pAkk or drinking water, colitis was induced by feeding mice with water containing 2.0% DSS for 7 days in IMQ-treated mice. Regular drinking water or pAkk supplement was provided until the end of the experiments. The comparison groups were given normal drinking water with IMQ or DSS treatment only.

Fig. 1.

Protocol of study design, and skin thickness change following IMQ application in mice with the Akk supplements or DSS treatment. (A) Protocol for IMQ plus DSS-induced colitis study, with or without pAkk supplementation. Mice receiving antibiotics for two days (ciprofloxacin (0.2 g/L) + Metronidazole (1 g/L) in drinking water) before experiments. After a 14-day pretreatment of oral pAkk or drinking water, colitis was induced by feeding mice with water containing 2.0% DSS for 7 days in IMQ-treated mice. Regular drinking water or pAkk supplement was provided until the end of the experiments. An intestinal permeability test was done before being sacrificed. Abbreviations: pAkk, pasteurized Akkermansia muciniphila; DSS, dextran-sodium sulfate; IMQ, imiquimod. (B) Akk supplements decreased skin thickness by IMQ or IMQ plus DSS treatments. The immunohistochemical analyses of (C) right ear and (D) back skin from different treatment groups demonstrated consistent results. (E) The skin thickness change of the right ear and (F) the back skin epidermis was calculated using Image J software. (G) splenic weight among various treatment groups. Abbreviations: IMQ, imiquimod; Akk, Akkermansia muciniphila; DSS, dextran sulfate sodium. *The analyses of skin thickness and spleen weight alterations were based on three times repeated experiments

The eight experimental groups included (1) control group (n = 5); (2) DSS alone group (n = 5); (3) IMQ group (n = 5); (4) IMQ plus DSS treatment group (n = 5); (5) pAkk alone group (n = 5); (6) pAkk supplements plus DSS(n = 5); (7) IMQ with pAkk supplement, no DSS treatment group (n = 5); (8) IMQ with pAkk plus DSS treatment group (n = 5). The results of skin changes and intestinal permeability tests were presented in Supplementary Fig. 2. Given the medium effect size of 0.25, with a power of 80%, α = 0.05, for eight groups, the sample size using one-way ANOVA is estimated to be 10. We then repeated the experiments to increase the power.

The intestinal permeability was examined as described [20]. Mice were orally gavaged with 150 μl of 100 mg/mL 4 kDa FITC dextran-labeled dextran beads (Sigma-Aldrich, 46,944) in PBS 4 h before euthanasia. FITC-derived fluorescence was quantified in the serum using a microplate luminometer EnSpire/PerkinElmer). Concentrations were determined using a standard curve generated by serial dilution of FITC-dextran.

Skin Thickness, Body Weight, and Tissue Weight Measurements

To avoid investigator bias, we measured the thickness of the right ear with a digital caliper daily during the seven days of IMQ and DSS treatment with or without pAkk pretreatment. Skin samples from the ear and back, as well as samples of spleen, liver, and fat, were collected at the end of the experiment and fixed in 10% formaldehyde for hematoxylin and eosin staining (Sigma, Germany, Leica Autostainer XL ST5010), anti-IL17A antibody (Ab79056, Abcam Limited, UK) or TNF-α (RM1005, Abcam Limited, UK). Each skin section was measured at three random spots, and the average skin thickness was calculated. We calculated the thicknesses of the epidermis and dermis of the different treatment groups using Image J software. (National Institute of Health, NIH).

Immunohistochemical Assessment of Intestine

After euthanasia, the mice ileums and colon were obtained to assess the histological inflammation. Digital photographs were taken, and the length of the colon was measured. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histological evaluation. Two of our authors, blinded to the experimental groups, assessed the histological sum scores of the ileum and colon based on the degree of inflammation, ranging from 0 (no inflammation) to 4 (severe inflammation). The average of the sum of the inflammation score in both the ileum and the colon was calculated.

Serum Cytokine Measurement

Serum samples were collected after the experiments. The cytokine levels in mouse serum (20 × diluted) were measured using a Multiplex cytokine bead array assay with the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTMAG-70 KPX32; Millipore). All measurements were performed on a MAGPIX instrument (Luminex, Austin, TX) following the manufacturer’s instructions, and data were acquired with xPonent software (Luminex). Statistical significance between treatment groups was assessed using the Mann–Whitney U test, with results expressed as mean ± SEM. A p-value of < 0.05 was considered statistically significant. Additional adjustments for multiple comparisons were made using the Benjamin-Hochberg false discovery rate (FDR) correction.

Stool Sample Collection, DNA Extraction, Library Construction, and Metagenomic Sequencing

Stool samples were collected and examined after experiments. Stool samples were obtained from each mouse after physical restraints, directly from the rectal aperture to avoid contamination. An Eppendorf tube pre-filled with Inhibitex Buffer, provided in the QIAamp Fast DNA Stool Mini Kit (Qiagen), was used to collect and stabilize the fecal specimen. The collection tube was carefully inserted into the rectal opening, ensuring minimal distress to the tissue. The sample was then secured in the tube, which was subsequently sealed and labeled with the appropriate identification details. Samples were stored at −20 °C until DNA extraction was performed. The collected fecal sample (about 200 mg) was used for total bacterial DNA extraction with QIAamp Fast DNA Stool Mini Kit (Qiagen, MD, USA) according to the manufacturer’s instructions. The quality and quantity of DNA were determined with NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE, USA) and stored at –80 °C before library construction and sequencing.

Extracted DNA (about 500 ng) was fragmented to approximately 350 base pairs by the Covaris S2 system (Covaris, Inc., Woburn, MA, USA) and then subjected to library construction with the Illumina DNA Prep Kit (Illumina, San Diego, CA). Sequencing was performed using an Illumina NovaSeq 6000 platform, resulting in paired-end (PE) reads of 150 bp in length.

Metagenomic Raw Read Processing

On a per-sample basis, raw read quality control was performed using the Kneaddata pipeline (https://github.com/biobakery/kneaddata), which integrates Trimmomatic [21] for trimming Illumina adaptors and low-quality regions, filtering short reads, and Bowtie2 [22] for identifying and removing host contaminations from human hg38 build (or mouse mm10 build) and PhiX genome. Trimmomatic options include “ILLUMINACLIP:NexteraPE-PE.fa:2:30:10” (identifying and removing adapters), “SLIDINGWINDOW:4:20” (trimming low quality region) and “MINLEN:50” (discarding reads shorter than 50 base pairs). After that, reads with low-complexity region and repeated sequences were identified and removed using the Komplexity software (https://github.com/eclarke/komplexity) with default settings.

Metagenome Assembly, Annotation, and Binning

Metagenome assembly was performed using MEGAHIT [23] to assemble the clean reads into contigs. As for taxonomy identification, two approaches were deployed: (1) read-based taxonomy, which analyzes QC-passed reads using Sourmash [24] to estimate relative abundance of taxa based on GTDB taxonomy [25], and (2) contig-based taxonomy, which predicts taxonomy of contigs using MMSeqs2 [25, 26] easy-taxonomy pipeline and estimates abundance by contigs depth, which is derived by the jgi_summarize_bam_contig_depths script analyzing bowtie2 alignment that maps the clean reads onto contig.

Open reading frame (ORF) prediction and functional annotation [including the Clusters of Orthologous Groups (COGs) family and Enzyme Commission (EC) number assignment] was performed by subjecting contigs into Prokka [27]. ORFs were also analyzed to identify corresponding KEGG Orthology (KO) number, Carbohydrate Active enZyme (CAZy) family, antibiotics gene, virulence factor using MMSeqs2 easy-search pipeline. MinPath [28] was used for pathway reconstruction by analyzing KO (for KEGG pathway) and EC (for MetaCyc pathway)²⁹ profiles of each sample. The abundance of gene families (or categories) was estimated by accumulating ORF depths, which is calculated by the tpm_table python script (https://github.com/EnvGen/toolbox) based on the number of unique reads mapped on each ORF, and presented in units of transcript per million (TPM). [30]

Metagenomics binning was performed by MetaBAT [31] to cluster contigs into genome “bins” [i.e., metagenome-assembled genomes (MAGs)]. The quality is assessed and taxonomy identified by CheckM lineage-specific workflow [32]. In addition, the taxonomy of MAGs is voted using the contig-based taxonomy of binned contigs, which extends the CheckM-predicted taxonomy to further depth in most cases.

Statistical Analysis, Bioinformatics Analysis and Microbial Gene Function Prediction

GraphPad Prism software 10 (GraphPad Software Inc., San Diego, CA, USA) was used to analyze experimental data and clinical data. One-way analysis of variance was used for the inter-group comparison. P < 0.05 was statistically significant. Other statistical analyses were performed using R (http://www.r-project.org/), unless otherwise specified. The read- or contig-based taxonomic profiles are imported and handled by the R package phyloseq [33] and processed for alpha diversity estimation. Beta diversity is analyzed and visualized by principal coordinate analysis (PcoA) via the R package ade4 [34] based on Bray–Curtis distance of species-level relative abundance profile. As for functional profiles, alpha diversity is represented in the number of observed entities. Beta diversity is also analyzed by PcoA based on Bray–Curtis distance of functional genes, families, or categories relative abundance profile.

MetagenomeSeq [35] is a method to determine features (operational taxonomic unit, or species) that are differentially abundant between two or more groups of multiple samples. It implements a novel normalization method, the cumulative sum scaling (CSS) normalization, to control for bias in measurements across taxonomic features and applies a zero-inflated Gaussian (ZIG) distribution to account for biases in differential abundance testing resulting from under-sampling of a microbial community.

The abundances of various gene families of experimental and control mice were scaled by total sum per sample and subjected to enrichment analysis of two-group comparison using several tests, including the Wilcoxon signed rank test, the ANOVA rank test, or the Kruskal–Wallis test with a Benjamin-Hochberg FDR correction to adjust p-values for multiple testing.

The bioinformatics analyses mentioned above were carried out by Germark Biotechnology Co., Ltd. (Taichung, Taiwan).

Results

pAkk Ameliorates Skin Thickening, Restores the Changes Of Splenic and Fat Weight and Serum Cytokines in IMQ-Induced Psoriasis Inflammation Mouse Model

We first asked if pretreatment with pAkk can inhibit the psoriatic inflammation in IMQ-induced mice model. pAkk has been linked to benefits in metabolic diseases and IBD by maintaining intestinal homeostasis and anti-inflammatory properties [36]. Mice were orally gavaged with 1.25 × 10 [9] colony-forming units (CFUs) of pAkk for 100 μL or drinking water alone, 2 weeks before the experiment. On day 0, these mice were subjected to topical IMQ, topical IMQ and DSS treatment, or placebo for 7 days. (Fig. 1A) pAkk significantly reduced the ear skin thickening and skin erythema during the 7 days of IMQ application or IMQ with DSS treatment. (Fig. 1B, Supplementary Fig. 2 and Supplementary Fig. 3 A). The H&E stain analysis on the IMQ-treated ear and back skin showed significantly reduced skin thickness gain after pAkk pretreatment, and the reduction was most significant in the epidermis. (Fig. 1C-F).

Next, we examined whether the protective effect of pAkk in reducing skin inflammation is linked to the inhibition of systemic pro-inflammatory cytokines after administering either IMQ or DSS. IMQ notably increased systemic pro-inflammatory cytokines by raising splenic weight. The splenic weight was higher after DSS stimulation in IMQ-treated mice. pAkk significantly decreased splenic weight gain in IMQ-treated mice subjected to DSS stimulation, although the reduction was not statistically significant compared to mice receiving IMQ alone. (Fig. 1G, Supplementary Fig. 3B) Mice treated with IMQ typically experienced sustained body weight loss. pAkk supplementation significantly prevented this IMQ-related weight loss. Similar trends were seen in fat weight changes, but not in the liver. (Supplementary Fig. 3C-3E).

Serum samples were collected at the end of the experiments. IMQ increased serum IL-17A levels compared to those of the controls. pAkk reduced circulating IL-17A in IMQ mice and IMQ plus DSS mice, although not significantly after FDR correction. pAkk supplementation also significantly lowered levels of TNF-α, IL-6, and G-CSF, especially in IMQ mice with DSS colitis. (Fig. 2A-C, Supplementary Fig. 4 A) The histological expression of IL-17A and TNF-α was also increased in IMQ mice and decreased in mice receiving the pAkk supplement. (Supplementary Fig. 4B, 4C).

Fig. 2.

Alterations of serum cytokines after Akk supplements in IMQ mice. Reduced serum (A) IL-17A, (B) TNF-α, (C) G-CSF levels in IMQ mice following Akk supplements. Abbreviations: Akk, Akkermansia muciniphila; DSS, dextran sulfate sodium; G-CSF, granulocyte colony-stimulating factor; IL-17A, interleukin-1A; IMQ, imiquimod; TNF-α, tumor necrosis factor-α

pAkk Restores the Intestinal Barrier Disruption and DSS-Induced Colitis in IMQ-Mice

The intestinal mucosal barrier plays an important role in maintaining intestinal homeostasis. Intestinal barrier dysfunction has reported to contribute to the transition of luminal contents into circulation, thereby inducing inflammation and activating the immune response. IMQ-treated mice receiving 7-day DSS presented acute illness, severe bloody diarrhea and colon length shortening. (Fig. 3A, 3B) We first investigated whether pAkk supplementation affects the intestinal permeability in IMQ-treated mice by using FITC-dextran administration. We then investigated if pAkk restores the colitis symptoms and colon length in IMQ-treated mice subjected to DSS administration.

Fig. 3.

Akk supplements restored intestinal length and reduced gut inflammation in mice receiving IMQ and IMQ plus DSS treatment. (A) Colon length and (B) pictures of the colon among treatment groups. (C, D) The H&E stain of the ileum and colon sections in treatment groups was presented. (E) The inflammation severity scores in the ileum and colon were summed. DSS, dextran sodium sulfate; IMQ, imiquimod

An increased intestinal permeability was found in mice both receiving IMQ alone and IMQ with DSS treatment. Mice receiving pAkk supplements have restored enhanced intestinal permeability compared to those with topical IMQ alone. (Supplementary Fig. 5 A) We excised the ilium and colon tissue for further analysis. Mice receiving DSS treatment had a significant colon shortening compared to controls. pAkk significantly restored the colon length shortening caused by DSS treatment (Fig. 3A, 3B). pAkk also ameliorated the inflammation of the ileum and colon mucosa in IMQ mice receiving DSS treatment. (Fig. 3C-E) One independent pathologist and one of our authors examined the degree of colitis inflammation in the ileum and colon sections. (Supplementary Fig. 5B and 5 C).

pAkk Supplement was Associated with an Altered Gut Microbiota

We finally analyzed the composition, abundance, and function of gut microbiota in fecal samples of mice after pAkk pretreatment using shotgun metagenomic sequencing. We performed metagenomic sequencing following these procedures: de novo assembly, prediction of open reading frames (ORFs), clustering and annotation of ORFs, and read mapping to assembled contigs. Using the taxa analysis results, we used the gene set enrichment analysis to compare the gut microbial profiles among various treatment groups.

To investigate whether pAkk supplement modulates the structure and composition of intestinal microbiota in IMQ-treated mice, the Wilcoxon rank sum test at three different taxonomic levels was used to compare the distribution of bacteria among experiment groups. Inter-group comparisons were made between IMQ alone and IMQ plus pAkk, as well as between PBS and IMQ alone. IMQ mice receiving pAkk had a significantly enhanced richness and diversity in alpha diversity analysis, compared to IMQ mice alone. (Fig. 4A).

Fig. 4.

The fecal microbiota alterations after Akk supplements in IMQ or IMQ plus DSS-colitis mice. (A) The α-diversity comparison of fecal microbiota between IMQ mice receiving Akk supplements or not. Akk enhanced the fecal microbial richness in IMQ mice. (B) Significant variations in the β-diversity of fecal microbiota in IMQ-treated mice with Akk supplements compared to those without Akk. Between-group inertia percentages were tested by the Monte-Carlo test (with 10,000 permutations) using the Bray–Curtis method. P values less than 0.05 were considered significant. (C) The discriminating microbial species on various treatments in IMQ mice were calculated by MetagenomeSeq [35]. IMQ-treated mice had a significantly reduced abundance of Bifidobacterium pseudolongum, Desulfovibrio genus, Ruminococcus flavefaciens, Turibacter and Alisitpes ihumi than PBS placebo. (D) Akk supplements restores the fecal microbiota in IMQ treated mice to the placebo status. Akk supplement increased the abundance of Muribaculaceae bacterium isolate-104 (HZI), Bifidobacterium psuedolongum, Desulfovibrionaceae bacterium, Erysipelotrichaeceae bacterium, and Alistipes ihumi. (E, F) The discriminating metabolic signaling pathways after Akk supplements in IMQ mice. The functional inference study compares the IMQ-treated mice with Akk supplements or not based on the KEGG pathways. Akk supplements significantly enhanced the gene expression involving the Cholinergic synapse, atrazine degradation, cell cycle, MAPK pathway, catechol degradation, and GABA shunt

The relative abundance of fecal microbiota in the IMQ group and IMQ receiving pAkk supplement group are presented in Supplementary Table 1. A significantly different fecal microbial composition in IMQ mice receiving pAkk pretreatment was found in the PCoA analysis using the Bray–Curtis method compared to IMQ alone. (Fig. 4B) The most discriminating species between experimental groups was conducted using MetagenomeSeq method. Mice following IMQ treatment had a significantly lower relative abundance of Bifidobacteirum (B) pseudolongum, Desulfovibrio bacterium, Ruminococcus falvefaciens, Turibacter sp TS3 and Alistipes ihumi than placebo controls. (Fig. 4C) pAkk-treated mice, on the contrary, had a significantly higher relative abundance of Muribaculaceae bacterium isolate 104 (HZI), B. pseudolongum, Desulfovibrionaceae bacterium, Erysipelotrichaeceae bacterium and Alistipes ihumi than IMQ-alone mice, as presented in Fig. 4D. Briefly, pAkk supplement restored the fecal microbiota to the status observed in the placebo-controlled status.

pAkk Supplements were Associated with Cholinergic Synapse, Cell Cycle and MAPK Pathway

Functional annotation of the assembled genomes was conducted using Prokka, and metabolic pathways were reconstructed with HUMAnN2 (https://github.com/biobakery/humann) referencing multiple databases, including the MetaCys and KEGG databases. The genetic inference study indicated that gene expression involving gamma-aminobutyric acid (GABA) shunt, cholinergic synapse, cell cycle, and Mitogen-Activated Protein Kinase (MAPK) pathways were enriched after the pAkk supplement in IMQ-mice. (Fig. 4E, F).

Discussion

This study showed that pAkk supplementation was associated with reduced skin and systemic inflammation in IMQ-treated mice. pAkk also improved intestinal permeability and colitis in IMQ mice given DSS. The positive effects of pAkk on psoriasis-like skin, as well as circulating and intestinal inflammation, were associated with changes in fecal microbiota, including increases in Muribaculaceae, B. pseudolongum, Desulfovirionaceae, Erysipelotrichaceae, and Alistipes ihumi. Finally, pAkk supplementation enriched genes involved in cholinergic synapse, cell cycle, and MAPK pathways.

The protective effect of pAkk has been demonstrated in colitis and inflammatory bowel diseases via decreasing IL-6/STAT3,^36,37 and NLRP3 inflammasome activation [38]. In the present study, pAkk supplementation was found to decrease levels of pro-inflammatory cytokines, including TNF-α, IL-6, G-CSF, and IL-17A. These cytokines are associated with psoriasis, those with greater disease severity,^39–41 or those with cardiovascular or dyslipidemia comorbidities. [41, 42] pAkk has been demonstrated to improve cognitive deficit in Alzheimer’s mice [43] and steatohepatitis by reduced hepatic proinflammatory macrophages (M1) and γδT and γδT17 cells, which is characterized in non-alcoholic steatohepatitis (NASH) patients [44]. In addition to improving lipid and metabolic dysregulation, pAkk also improves cognitive function in aged mice by altering gut microbiota and reducing IL-6 expression in both serum and hippocampal tissue. [45] The results from previous studies, together with the current study, implied the anti-inflammatory effect of pAkk supplement may not only improve skin inflammation but also provide a beneficial effect in decreasing cardiovascular and metabolic comorbidities in psoriasis disease.

Our results showed that pAkk supplementation restored the fecal microbiota of IMQ mice to the status observed in the placebo-controlled status. (Fig. 4C, 4D) These findings provided evidence of the beneficial role of pAkk in reducing inflammation by altering the gut microbiota to a protective state. Among the pAkk-enhanced fecal microorganisms, B. pseudolongum has been shown to improve colitis symptoms by maintaining intestinal epithelial integrity [46] by activating PPAR/STAT3 pathways and increasing SCFA production. SCFAs are crucial in maintaining gut health, beneficial for intestinal and systemic inflammation, and alleviating psoriatic inflammation. [47] The newly identified species of Alistipes has been reported to co-occur with B. pseudolongum and produce acetate and propionate. [46, 48] However, its clinical implications in human diseases remain poorly characterized. [48] Desulfovibrionaceae is a genus of sulfate-reducing bacteria widely explored in several human diseases, including IBD, metabolic syndrome, neurogenerative, or cardiovascular diseases. [49] Muribaculaceae has been reduced in colitis mice models [50]. On the contrary, Erysipelotrichaceae is increased in mice models of anxiety, autism, and colitis. [50–52] The clinical implications of these microorganisms remain to be explored in humans.

The current study further highlighted the enriched gene expression involving multiple metabolic pathways (Fig. 4E, 4F) in pAkk-supplemented IMQ mice. The imbalance of GABA is associated with neurologic and psychological disorders such as Alzheimer’s disease, Parkinson’s disease, depression, anxiety, and stress. [53] GABA has been recently proposed as a potential postbiotic neurotransmitter in the gut-brain skin axis linking psoriasis and mental health. [54] A dysregulation of cholinergic signaling has been associated with autoimmune diseases, such as RA, SLE, neurodegenerative diseases [55] and psoriasis [56]. Psoriasis patients tend to have a higher acetylcholine (Ach) level, especially in those with longer disease durations, and greater body mass index (BMI). [56] Cholinergic synapses are crucial for memory, learning, attention, and higher brain functions. Their ubiquity in the human central nervous system underscores the importance of cholinergic transmission in cognitive processes and age-related cognitive decline, including dementias such as Alzheimer's disease. [57] The beneficial effect of pAkk supplement in modulating metabolic pathways involving psoriasis and neurogenerative comorbidities may be worth further exploration.

We observed an involvement of gene expression of the MAPK pathway in Akk-supplemented IMQ-mice. Activation of MAPK pathways has been associated with cancer, cardiovascular, neurological, metabolic, and inflammatory disorders, including psoriasis and colitis. [58–60] Campylobacter jejuni (C. jejuni) induced colitis mainly by activating the PI3K-AKT and MAPK signaling pathway, and fecal microbial transplantation (FMT) has effectively reduced C. jejuni-induced colitis [61]. pAkk, the core flora of FMT, along with its microbial metabolites butyric acid and deoxycholic acid, inhibit these signaling pathways, counteract C. jejuni infection and alleviate colitis. We postulated that the pAkk supplement might alleviate the inflammation of the skin and colon via modulating MAPK pathways.

The present study demonstrated that IMQ alone significantly causes gut dysbiosis and intestinal inflammation, leading to a shortened colon length. IMQ further aggravates the severity of DSS-induced colitis. Although DSS alone did not cause significant psoriasis inflammation in the present study, DSS has been documented to disrupt the skin barrier and increase transepidermal water loss via the activation of cholinergic signaling pathways. [62] These results proposed a two-hit hypothesis for colitis [19] development in IMQ-mice: psoriasis-induced disrupted gut homeostasis and a secondary environmental challenge. The results from our colitis experiments demonstrated that pAkk restored intestinal epithelial integrity and improved DSS-induced colitis in IMQ mice (Fig. 5), probably through the modulation of the Th17 and cholinergic signaling pathways. This supports the idea that targeting gut microbiota can be an effective strategy for managing psoriasis-like inflammation via the gut-skin connection.

Fig. 5.

The proposed mechanisms of Akkermansia muciniphila in ameliorating the psoriasis-like inflammation via modulating gut microbiota

The alterations of fecal microbiota may be associated with multiple factors. The age, sex, host genetic background, infection, use of antibiotics, probiotics or prebiotics, diet patterns, and habitat environment, such as cage factors, were all significant factors affecting the composition of fecal microbiota. Due to lacking germ-free laboratory conditions, we utilized short-term antibiotics [63] to prevent background microbiota signals before experiments. We ensured uniformity among the study mice by selecting mice of the same age, sex, and species. Additionally, the same experiment mice group were housed in a single cage, where they were provided with identical bedding materials throughout the experiment. This standardization was implemented to minimize variability and control for environmental factors. We aimed to avoid all background biases in interpreting experiment results.

There are several limitations in the present study. First, although antibiotics were administered before the pAkk supplement to mitigate background microbiota (Supplementary Fig. 6), in which very low or no DNA was detected after antibiotic exposure, the absence of germ-free mice limits our ability to draw definitive causal inferences regarding Akk’s effects. We adopted a short-term, 2-day antibiotic course to minimize antibiotic-induced immune bias; however, the potential nonspecific effects of antibiotics on immune cells and the intestinal barrier cannot be overlooked. All mouse groups received antibiotics beforehand, and no significant differences in intestinal permeability, colon length, or inflammatory profiles were observed between control and pAkk-treated groups. Second, this study was conducted with a limited number of B6 mice, and the findings may not be generalizable to other species or experimental designs, warranting further validation. Third, only male C57BL/6 mice were used to minimize hormonal variation, enhance inflammatory responses, and maintain consistency with most published models; however, this may introduce selection bias and limit generalizability. To reduce investigator bias, ear skin thickness in different treatment groups was measured with a digital caliper, while inflammatory grading of intestinal histological sections was independently reviewed by two blinded investigators. For skin thickness evaluation, three randomly selected spots per section were measured using ImageJ, and the mean value was calculated. We did not examine different doses of pAkk treatment, precluding assessment of dose–response relationships. Furthermore, fecal and circulatory metabolite levels were not measured; thus, the metabolic pathways implicated by gene enrichment analysis in psoriasis-like mice require further investigation.

Finally, this study employed the acute IMQ mouse model, which reproduces key inflammatory pathways observed in human psoriasis. However, as it reflects only short-term skin inflammation, the results cannot be directly extrapolated to the long-term course of chronic psoriasis in humans. While the model offers valuable mechanistic insights, its inherent limitations, including short disease duration, differences in microbiota between mice and humans, and potential variations in administration methods for probiotics, should be addressed in future studies to enhance clinical translatability.

In conclusion, pAkk may help reduce both skin and systemic inflammation in the IMQ-treated mice and the DSS-induced colitis mouse model. Future studies should incorporate chronic or humanized models and optimize delivery strategies to reflect the human context better.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Akk

Akkermansia muciniphila

COG

Clusters of Orthologous Groups

DSS

Dextran sodium sulfate

EC

Enzyme commission

FDR

False discovery rate

FMT

Fecal microbial transplantation

IBD

Inflammatory bowel disease

IBS

Irritable bowel syndrome

KO

KEGG orthology

IMQ

Imiquimod

MAG

Metagenome-assembled genome

ORF

Open reading frame

OTU

Operational taxonomic units

pAkk

Pasteurized Akkermansia muciniphila

PCoA

Principal coordinate analysis

PsA

Psoriatic arthritis

RA

Rheumatoid arthritis

SCFAs

Short-chain fatty acids

SLE

Systemic lupus erythematosus

TPM

Transcript per million

Author Contributions

YC, CW, and HJH had full access to all the data in the study and took responsibility for the integrity and accuracy of the data analysis. YC and CW conducted all study conception and design. YC, CT, and CW performed the first drafting of the manuscript. YFC, and JS conducted and supervised the animal experiments. YFC, HJH, and CT performed the statistical analysis. All authors contributed to the acquisition, analysis, and interpretation of the data. All authors read and approved the manuscript.

Funding

This work was supported by grants NSTC 108–2314-B-075A-008 and 110–2314-B-075A-008 and TCVGH-1136801B.

Data Availability

Raw sequencing data files of experimental samples have been deposited in NCBI affiliated with BioProject PRJNA1171501. (will be released one year later, and the reviewer’s link at https://dataview.ncbi.nlm.nih.gov/object/PRJNA1171501?reviewer=mgtdqlr9ckuh6lio6ddh9k4b4n in read-only format.)

Declarations

Ethical Approval and Consent to Participate

The experiments were carried out in accordance with the protocols approved by the IACUC Taichung Veterans General Hospital, La-1091737. The present study followed national guidelines of the 3Rs for humane animal treatment and complied with relevant legislation from the Ministry of Agriculture, Taiwan. The study is reported in accordance with ARRIVE guidelines.

Consent for Publication

Not applicable.

Competing interests

The authors declare no competing interests.