Age-Dependent Alterations in Intestinal Barrier Function: Involvement of Microbiota and TLR4 Signaling

Simple Summary

The intestinal barrier, a crucial defense system against pathogens and toxins, undergoes dynamic changes throughout a lifespan. While age-related gut decline and systemic inflammation are well-documented, a systematic understanding of how the gut microbiota and host immune signaling coordinately evolve from infancy to old age remains limited. This study comprehensively investigated these interactions across major developmental stages, including pups, adults, middle-aged, and old age in mouse models. It was found that microbial diversity and beneficial metabolites peak in adulthood, while inflammatory signals progressively increase with aging. Crucially, the immune receptor Toll-like receptor 4 (TLR4) was identified as a key driver of age-related gut deterioration. These findings reveal that aging disrupts the delicate host–microbe dialogue, leading to barrier dysfunction and chronic inflammation. Targeting this interaction, especially through TLR4 modulation, presents a promising strategy to support gut health and promote healthier aging.

Abstract

The intestinal barrier undergoes profound changes with age, impacting local immunity and systemic health, yet the mechanisms coordinating immune and microbial dynamics across the lifespan remain incompletely understood. Toll-like receptor 4 (TLR4) serves as a key mediator of host–microbiota interactions. This study investigated age-related changes in barrier function and the role of TLR4 using C57BL/6J and TLR4 knockout (TLR4^−/−) mice across key developmental stages: pups (postnatal day 9), adults (2–4 months), middle-aged (7–9 months), and old (16–19 months). Through a multi-layered approach integrating histology, microbiome profiling, short-chain fatty acid (SCFA) analysis, cytokine quantification, ex vivo functional assays, and transcriptomics, we identified a multi-phase process of intestinal remodeling. Pup-P9 mice exhibited immature colonic structure, a simple microbiota dominated by Firmicutes and Proteobacteria, and undetectable acetic acid level. Adults reached peak diversity and SCFA concentrations, marked by a rise in Bacteroidota and the emergence of Akkermansia. In middle and old age, pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased, Bacteroidota declined while Firmicutes, Actinobacteria, and Turicibacter expanded, and aged colons showed blunted ex vivo responses to IL-1β. This age-associated functional decline phenotype was absent in TLR4^−/− mice, supporting the involvement of TLR4 signaling. Transcriptomics further revealed biphasic PI3K/Akt activation in both pups-P9 and old mice. Together, these findings suggest a systemic rewiring of host metabolic and immune signaling pathways in response to an aging microbiota, highlighting this dynamic, lifespan-wide microbiota–host signaling axis as a potential intervention target.

1. Introduction

The intestinal barrier, a dynamic physiological system composed of biological, chemical, mechanical, and immune components, undergoes continuous adaptation throughout the lifespan in response to age and microbial cues [1,2]. From birth to senescence, barrier function evolves significantly, intimately linked to microbiota colonization, immune maturation, and environmental exposure [3,4]. In early life, the barrier is underdeveloped, exhibiting high permeability and immature tight junctions. While this facilitates nutrient absorption for rapid growth, it also increases vulnerability to microbial invasion, particularly in premature or immunocompromised neonates [5,6]. By adulthood, the barrier typically achieves relative stability, supported by robust tight junction complexes, a resilient microbiota, and refined immune responses [7]. However, with advancing age, this ecosystem deteriorates: mucosal integrity declines, microbial dysbiosis emerges, and immune responsiveness wanes, collectively predisposing individuals to metabolic and immune disorders [8,9]. In the elderly, barrier dysfunction is closely linked to “inflammaging”, a chronic low-grade inflammatory state driven largely by the translocation of microbial products such as lipopolysaccharide (LPS) into circulation [10]. This process establishes a self-perpetuating cycle of inflammation and barrier disruption [11]. Notably, the absence of age-related pro-inflammatory factor accumulation in germ-free animals underscores the pivotal role of the microbiota in driving this phenomenon [12]. Therefore, a systematic investigation spanning key developmental windows, from infancy to senescence, is essential to delineate how age-dependent microbial and immunological dynamics collectively shape intestinal barrier function.

Toll-like receptor 4 (TLR4), a pattern recognition receptor for LPS, plays a significant role in intestinal immunity, contributing to both homeostasis under physiological conditions and inflammation upon overactivation [13,14]. Its involvement in the pathogenesis of inflammatory bowel disease (IBD) highlights its potential as a therapeutic target at the interface between the microbiota and host immunity [15,16,17]. Beyond its established roles in early immune and microbial development, TLR4 has recently emerged as an important mediator in aging-related inflammation and barrier dysfunction [18]. However, TLR4 operates within a broader network of pattern recognition receptors and signaling pathways (such as nucleotide-binding oligomerization domain (NOD)-like receptors) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) (also involved) that collectively maintain intestinal immune homeostasis [14]. Elucidating how age-dependent microbial dynamics specifically engage TLR4 signaling and how this interaction differs across life stages is thus valuable for understanding the mechanisms underlying age-associated gastrointestinal and systemic disorders.

Despite growing interest in age-related intestinal changes, few studies have systematically compared barrier function, microbial composition, and immune signaling across the full lifespan [4]. Most existing research has focused on either early development or late senescence [2,4,6], leaving the causal relationship between age-related microbial shifts and intestinal inflammation incompletely understood. We hypothesized that age-associated intestinal decline results from coordinated, stage-specific changes in gut microbiota and host immune signaling, with TLR4 playing a key modulatory role. To test this, we comprehensively evaluated intestinal barrier function across four developmental stages (pup, adult, middle-aged, and old) in wild-type and TLR4 knockout mice, integrating analyses of microbiota composition, short-chain fatty acid profiles, transcriptomic data, and systemic immune responses. By delineating how host–microbe interactions evolve with age, this study aims to provide an integrated mechanistic framework and identify potential targets for preserving gut health.

2. Materials and Methods

2.1. Chemical Reagents

The total RNA extraction kit was obtained from Proteinssci Biotech (Shanghai, China). ChamQ Universal SYBR Green qPCR Master Mix, enhanced chemiluminescence (ECL) kit, and reverse transcription premix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit, poly-vinylidene fluoride (PVDF) membranes, and RIPA lysis buffer were supplied by Beyotime Biotechnology Institute (Shanghai, China). Fetal bovine serum (FBS), phenol red-free Opti-MEM medium, and phosphate-buffered saline (PBS) were acquired from Gibco Company (Grand Island, NY, USA). Bovine serum albumin (BSA), hydrocortisone, and penicillin-streptomycin solution were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Recombinant human interleukin-1beta (IL-1β) was sourced from PeproTech Company (Cranbury, NJ, USA), while the TGuide S96 magnetic bead-based kit was from TIANGEN (Beijing, China). All chemical reagents used in this study were of analytical grade.

2.2. Antibodies

Rabbit polyclonal primary antibodies targeting PI3K, phosphorylated PI3K (pPI3K) and β-actin were acquired from Abcam (Oakland, CA, USA). Rabbit polyclonal primary antibody against Akt and goat anti-rabbit secondary antibody were obtained from STATER (Hangzhou, China). Rabbit polyclonal primary antibodies against pAkt were purchased from Cell Signaling Technology (Danvers, MA, USA). All primary antibodies were applied at a dilution of 1:1000, while the secondary antibodies were used at 1:5000.

2.3. Animals and Treatments

This study utilized C57BL/6J wide-type (WT) mice and TLR4 gene knockout (TLR4^−/−) mice across different age groups. Pregnant WT mice and breeding pairs of TLR4^−/− mice were purchased from GemPharmatech (Nanjing, China). The offspring were bred and maintained until reaching the following age stages: postnatal day 9 (pup-P9, PW for WT mice and PK for TLR4^−/− mice), 2–4 months (adult, AW for WT mice and AK for TLR4^−/− mice), 7–9 months (middle-age, MW for WT mice and MK for TLR4^−/− mice), and 16–19 months (old, OW for WT mice and OK for TLR4^−/− mice). Animals were used without sex selection. At the pup stage, litters were treated as experimental units without sex differentiation, as reliable visual sex identification was not feasible. This mixed-sex approach was maintained across all other age groups to ensure methodological consistency. Although pups were derived from multiple litters and randomly assigned to minimize potential litter effects, formal statistical modeling to account for litter-specific variability was not performed. Animals were randomly sorted and grouped using computer-generated random numbers. The testing sequence for all samples was fully randomized. All animals were housed under a 12 h/12 h light–dark cycle at a controlled temperature of 25 ± 1 °C in a specific pathogen-free (SPF) animal facility, with free access to deionized water and standard rodent chow. All gavage procedures were performed by well-trained personnel using gentle and swift techniques to minimize animal stress. Mice were euthanized at the designated time points; euthanasia was carried out by carbon dioxide inhalation, followed by cervical dislocation as secondary confirmation to ensure a painless death, and colon tissues along with luminal contents were collected and stored at −80 °C for subsequent analysis. All experimental protocols were reviewed and approved by the Animal Experimentation Ethics Committee of Jiangsu University (Approval No. UJS-IACUC-2021040703).

2.4. Ex Vivo Inflammatory Colon Model

An ex vivo colon culture model was adapted from a previously established protocol with slight modifications [19]. The culture medium consisted of phenol red-free Opti MEM supplemented with 10% FBS, 2 mM L glutamine, 0.2 U/mL insulin, 20 ng/mL epidermal growth factor, 200 nM hydrocortisone, and 1% penicillin streptomycin. Colons were pre-incubated with this medium for 1 h at 37 °C, followed by stimulation with 1 ng/mL recombinant mouse IL-1β for 2 h to induce inflammation. After treatment, tissues were collected, snap-frozen, and stored at −80 °C for subsequent RNA isolation.

2.5. Hematoxylin-Eosin Staining

Colon tissues were processed for histopathological assessment. Briefly, tissues were fixed in 4% paraformaldehyde for 24 h, followed by paraffin embedding and sectioning at a thickness of 4 μm. Tissue sections were then stained with hematoxylin and eosin (H&E). Histological evaluation and image acquisition were conducted using a Nikon Eclipse MA200 microscope (Nikon Instruments Inc., New York, NY, USA) at ×200 magnification.

2.6. Gut Microbiota Analysis

Microbial community profiling was performed by 16S rDNA gene sequencing and bioinformatics analysis, which were conducted by Biomarker Technologies Co., Ltd. (Beijing, China). In brief, total microbial DNA was extracted from colon content samples using the TGuide S96 magnetic bead-based kit. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified by PCR with the primers 338F and 806R on an ABI GeneAmp^® 9700 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation and sequencing were carried out on the Illumina NovaSeq platform, generating 150 bp paired-end reads. A total of 48 samples were sequenced in the same run, including colon content samples from the present study and samples from other concurrent experiments. After quality filtering, adapter trimming, and read merging, all samples met the minimum sequencing depth requirement of 41,263 clean reads per sample, with an average depth of 75,060 clean reads per sample. Rarefaction curves confirmed that this depth was sufficient to capture the majority of microbial diversity. To ensure comparability across samples, sequencing depth was normalized by rarefying to the minimum read count prior to downstream diversity analyses. High-quality reads were then clustered into operational taxonomic units (OTUs) at a 97% similarity threshold. Taxonomic classification was performed using the Ribosomal Database Project (RDP) reference database via the BMKCloud platform (www.biocloud.net, accessed on 20 April 2025.), and subsequent analyses were carried out based on the resulting OTU table. Phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) predicted metabolic pathways, annotated via Kyoto encyclopedia of genes and genomes (KEGG).

2.7. Quantification of Short-Chain Fatty Acids (SCFAs)

The contents of SCFAs, including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid, were measured using gas chromatography (GC) following our previously established protocol [20]. Briefly, 100 mg of colonic contents were homogenized in PBS, acidified with 75 μL of H₃PO₄, and centrifuged. The supernatant was then filtered through an organic-phase membrane and further analyzed on a Nexis GC-2030 system (Shimadzu, Kyoto, Japan) equipped with a DB-FFAP column (30 m × 0.25 mm × 0.25 μm). Chromatographic separation was achieved under the following temperature program: the oven was held at 100 °C for 5 min, then increased at 10 °C/min to 150 °C, and maintained at this temperature for 12 min. Nitrogen was used as the carrier gas. The quantification was performed using the relative standard curve of external standards, expressed as mmol/L.

2.8. Quantitative Reverse Transcription PCR (qRT-PCR)

Total RNA was isolated from colons using a high-purity total RNA extraction kit, and its concentration and purity were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized from 2 μg of total RNA using reverse transcription premix. Quantitative RT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using ChamQ Universal SYBR Green qPCR Master Mix. All primer sequences are listed in Table S1 with Gapdh serving as the endogenous reference gene. Relative mRNA expression levels were calculated using the 2^−ΔΔCt method and presented as fold changes normalized to the adult/control group.

2.9. Transcriptomic Analysis

Transcriptomic sequencing was performed on colonic tissues from mice across different age groups. Total RNA extraction was carried out as described in the qRT-PCR section above. Libraries were prepared from 1 μg RNA per sample using the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit (Yeasen Biotechnology, Shanghai, China) and sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Raw reads were processed to obtain clean reads by removing adapter sequences, poly-N, and low-quality reads. Clean reads were mapped to the mouse reference genome (GRCm38) using Hisat2. Gene expression levels were quantified using FeatureCounts and normalized as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed gene (DEG) analysis was performed using DESeq2 in R, applying thresholds of adjusted p < 0.05 and |log₂FC| ≥ 1. Gene ontology (GO) enrichment analysis was conducted using clusterProfiler with Wallenius correction, and KEGG pathway enrichment analysis was performed. Volcano plots were generated to visualize the differential expression results. All sequencing and primary bioinformatic procedures were completed by Beijing Biomarker Technologies Co., Ltd. (Beijing, China).

2.10. Western Blot Analysis

Total protein was extracted from colons using RIPA lysis buffer, and protein concentration was determined with a BCA protein assay kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto PVDF membranes. After blocking with 3% BSA or 5% non-fat milk for 1 h, membranes were washed and incubated overnight at 4 °C with primary antibodies against Akt, pAkt, PI3K, pPI3K, and β-actin, followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at room temperature for 1 h. β-Actin served as the internal loading control. Protein bands were detected using an ECL kit and visualized with an ImageQuant™ LAS 4000 imaging system (GE Healthcare, Pittsburgh, PA, USA). Band intensities were quantified using ImageJ software (version 1.54p, National Institutes of Health, Bethesda, MD, USA).

2.11. Statistical Analysis

All data were presented as means ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA). For comparisons among multiple groups, using one way Analysis of Variance (ANOVA) followed by Tukey’s test or Kruskal–Wallis test with appropriate post hoc tests. Correlation analyses were performed in the R software environment (version 4.5.1, https://www.r-project.org/, accessed on 17 November 2025). A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Histopathological Evaluation of Colon Morphology Across Age Groups

Figure 1A schematically illustrates the experimental timeline and sampling time points. Representative H&E-stained colon sections at each stage are presented in Figure 1B–D. In pup-P9 mice, the colon exhibited a notably smaller lumen with fewer mucosal folds, limited crypt numbers, and a thinner intestinal wall. In contrast, colon architecture remained intact in adult, middle-aged, and old groups, characterized by well-organized crypts and the absence of evident inflammatory infiltration.

3.2. Age-Dependent Shifts in Colonic Microbial Diversity

Preliminary analysis revealed that colonic microbial abundance and diversity exhibited a positive correlation between age and microbial profiles from the pup-P9 to adult stage, followed by a decline from adulthood to old age. This trend was consistent across multiple alpha-diversity indices, including the ACE, Chao1, Shannon, and Simpson indices (Figure 2A–D). As illustrated in Figure 2E, Venn diagram analysis demonstrated distinct microbial compositional profiles among age groups. The number of unique OTUs was 557 in pups-P9, 873 in adults, 834 in middle-aged mice, and 642 in old mice, with only 9 OTUs shared across all groups, supporting age-dependent divergence in gut microbiota structure. Principal coordinate analysis (PCoA) further confirmed significant separation of microbial communities among different age groups, indicating notable differences in overall community similarity (Figure 2F).

3.3. Age-Dependent Alterations in Gut Microbiota Composition

Analysis of the microbial composition at the phylum and genus levels revealed marked differences across age groups, characterized by distinct, age-dependent abundance patterns of specific taxa. Overall microbial richness increased with advancing age, accompanied by marked structural reorganization.

At the phylum level, the PW group exhibited a simple microbial structure dominated by Firmicutes and Proteobacteria. In contrast, the AW group showed increased microbial diversity, characterized by a marked rise in Bacteroidota, a decrease in Proteobacteria, and the appearance of Actinobacteria, Desulfobacteriota, and Verrucomicrobia. The MW and OW groups displayed a composition distinct from that of the AW group, with a significant decrease in Bacteroidota and a notable increase in Firmicutes and Actinobacteria (Figure 3A). At the genus level, the PW group was dominated by Ligilactobacillus and Rodentibacter. The AW group also demonstrated the highest genus-level diversity, and its taxonomic composition was substantially different from that of others. In the MW and OW groups, the relative abundance of Dubosiella, Turicibacter, Limosilactobacillus, and Bifidobacterium increased markedly (Figure 3B).

Specific taxa exhibited clear age-dependent patterns: Ligilactobacillus was enriched in pups-P9, whereas unclassified_Muribaculaceae was more abundant in adults; Akkermansia was exclusively detected in the mature hosts (AW and MW), while it was not detected in pups-P9 and old mice (Figure 3C–E). Consistent with the observed differences in genus diversity and composition, linear discriminant analysis Effect Size (LEfSe) analysis further identified specific gut microbial biomarkers that varied across developmental stages (Figure S1A,B). Circular cladogram clearly demonstrates systematic, stage-specific successional patterns in microbial communities across all taxonomic levels (phylum, class, order, family, genus, species), which are closely associated with host age. The association analysis network visualized the most dynamically changing genera during development as Ligilactobacillus and Rodentibacter (Figure S1C).

These compositional shifts suggest functional reconfiguration of the gut ecosystem across the lifespan. The dominance of Firmicutes and Proteobacteria in pups-P9 reflects a community specialized for a milk-based diet, while the expansion of Bacteroidota and emergence of Akkermansia in adults provides support for a shift towards enhanced metabolic capacity [21]. In contrast, the decline of Bacteroidota and the enrichment of Actinobacteria and Turicibacter in middle-aged and old mice may contribute to a state of dysbiosis and low-grade inflammation, offering a potential mechanism for the increased inflammatory cytokine profile observed during aging [22].

3.4. PICRUSt2 Analysis

The functional annotation bar plot (Figure S2A) illustrates the relative abundance distribution of KEGG pathways across bacterial phyla. Overall, the category “Global and overview maps” consistently exceeded 60% abundance, underscoring that core metabolic and regulatory pathways form the foundational functional profile of the microbial community. Significant age-dependent functional variations were observed between groups (Figure S2B–G). The PW group exhibited elevated “Human Diseases” pathways compared to the AW group (Figure S2B, p < 0.05), and higher “Environmental Information Processing” versus the AW (Figure S2B, p < 0.05), MW, and OW groups (Figure S2C,D, p < 0.01). In contrast, AW displayed reduced “Human Diseases” and “Genetic Information Processing” compared to MW and OW groups (Figure S2E,F, p < 0.05). Additionally, the OW group was further enriched in “Human Diseases” and “Genetic Information Processing” but depleted in “Metabolism” pathways (Figure S2G, p < 0.001).

3.5. Age-Dependent Changes in Short-Chain Fatty Acids

The concentrations of SCFAs exhibited significant lifespan-associated changes, following a parabolic trajectory (Figure 4A). Specifically, total SCFA levels increased from early life through development into maturity (adulthood and midlife) and subsequently declined during aging (old age). Marked differences were observed in individual SCFAs, particularly between the pup-P9 stage and other age groups (Figure 4B–G, p < 0.05 for acetic acid and propionic acid). Notably, acetic acid was undetectable in pup-P9 mice (Figure 4B). Additionally, valeric acid was not detected in the adult and old groups. Correlation analysis suggested trends linking specific microbial taxa with SCFA concentrations. At the phylum level, acetic acid tended to correlate negatively with Bacteroidota, whereas butyric acid showed a potential positive correlation with Acidobacteriota (Figure 4H). At the genus level, Rodentibacter exhibited a possible negative correlation with acetic acid, while Desulfovibrio showed a potential positive correlation with iisobutyric acid (Figure 4I, p < 0.05).

The absence of acetic acid in pups-P9 is consistent with the dominance of Ligilactobacillus and Rodentibacter, both negatively correlated with this metabolite. This confirms that the early-life taxonomic configuration is optimized for milk digestion rather than fiber fermentation [23]. In adulthood, this functional configuration shifts, as the expansion of Bacteroidota and Muribaculaceae is mechanistically linked to peak SCFA production, reflecting a mature ecosystem adapted for complex polysaccharide metabolism [24].

3.6. Age-Dependent Changes in Gut Inflammation and Immune Responses

Gut inflammation and immune responses exhibited age-associated alterations. Our study demonstrated that the basal levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), IL-1β, and TLR4 increased in an age-dependent manner (Figure 5A–D). Using an ex vivo inflammatory colon model, we further compared the response of colon tissues from mice at different ages to IL-1β stimulation. The results showed that the pro-inflammatory response was most pronounced in pup-P9 mice (p < 0.001) and gradually declined with advancing age (Figure 5E–G), indicating that colon tissue in early life possesses a more sensitive and robust innate immune response to inflammatory stimuli, while the old group lost sensitivity to external stimuli (p > 0.05). However, in TLR4-knockout mice, IL-1β stimulation failed to elicit an immune response (Figure S3), suggesting that TLR4 is involved in mediating this inflammatory pathway. To further elucidate the role of gut microbiota in intestinal inflammation, we performed a targeted analysis of inflammation-associated microorganisms. Correlation analysis showed no significant associations at the phylum level (Figure 5H). At the genus level, both Turicibacter and Limosilactobacillus exhibited trends toward positive correlations with pro-inflammatory cytokine expression (Figure 5I, p < 0.05). The positive correlation between Turicibacter and pro-inflammatory cytokines, together with the enrichment of this genus in middle-aged and old mice, implicates this taxon in the age-associated pro-inflammatory shift. Interestingly, the positive correlation between Limosilactobacillus (a genus conventionally regarded as beneficial) and inflammation in older groups suggests that the aging host environment may fundamentally alter the functional role of commensal bacteria [25].

3.7. Functional Enrichment Analysis of DEGs via KEGG Pathway Mapping

To explore age-related alterations in genome-wide transcriptional profiles, we performed RNA-sequencing on full-thickness colonic samples. Volcano plot analysis demonstrated that aging significantly altered the gene expression profile in the colon, with a substantial number of differentially expressed genes (both up- and down-regulated) across age-related comparisons (Figure 6A–D). KEGG enrichment analysis of the differentially expressed genes revealed that the PI3K-Akt signaling pathway was consistently enriched in multiple pairwise comparisons, including between PW and AW, PW and MW, AW and MW, as well as AW and OW groups (Figure 6E–H). The recurrent identification of this pathway across distinct age transitions highlights its potential central role in colonic aging and associated pathophysiological changes.

To validate the transcriptomic findings regarding the PI3K-Akt signaling pathway, we assessed its activation at the protein level via Western blot analysis. The results confirmed that age significantly influenced the phosphorylation status of this pathway in colon tissue. Specifically, the ratios of p-PI3K/PI3K and p-Akt/Akt were markedly elevated in both the pup-P9 and old groups relative to the adult and middle-aged groups (Figure 7A–C). This biphasic activation pattern implies that colon tissue in early and late life stages may exist in a functionally susceptible state, which could predispose it to heightened inflammatory reactivity.

4. Discussion

Growth and development constitute key drivers of gut microbiota differentiation in mice. Numerous studies have documented significant differences in the gut microbiota of mice across different age stages. Reports indicate that pups harbor a less abundant microbial community, accompanied by dramatic shifts after weaning [26]. Similarly, aged mice exhibit distinct alterations in microbial composition and diversity [27]. Our study demonstrates that gut microbial composition, metabolic output, and host signaling pathways undergo stage-specific and coordinated changes throughout the lifespan, collectively shaping intestinal homeostasis in an age-dependent manner: progressing from an immature state in infancy, reaching peak functional capacity during adulthood, and gradually declining in later life. Together, these findings reveal a coordinated, stage-specific reconfiguration of the gut ecosystem: microbial composition shapes SCFA profiles, which in turn modulate host immune responses and intracellular signaling pathways (a schematic diagram summarizing lifespan-dependent host–microbiota–TLR4 interactions shown in Figure S4). This integrative view suggests age-associated intestinal decline not as isolated changes in any single component, but as a progressive dysregulation of the entire host–microbe interaction network, with fundamental implications for barrier function, immune regulation, and disease susceptibility across the lifespan.

Consistent with prior reports, we observed pronounced differences in gut microbiota structure across life stages. Bacteroidota are generally considered beneficial, whereas Proteobacteria are often considered a marker of dysbiosis [28]. The progressive increase in the ratio of Bacteroidota to Firmicutes (B/F) with developmental maturation reflects a dynamic restructuring of the microbial community [29], suggesting a shift in collective metabolic potential and enhanced resilience against dysbiosis in adult mice. A decreased B/F ratio with aging indicates that gut microbial community in old mice exhibits a general trend toward functional dysregulation. The increase in Bifidobacterium (belonging to Actinobacteria) in the middle-aged and old groups suggests that the gut environment in middle and old age may rely more heavily on beneficial Actinobacteria for regulation, given its established functions in aiding digestion, enhancing immunity, and exerting antioxidant effects. The negative correlation between the enrichment of beneficial genera, such as Ligilactobacillus, in pups-P9 and systemic pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) underscores their potential role in early-life immune priming and intestinal barrier maintenance. This correlation directly links the early-life microbial configuration to the low inflammatory tone observed in pups-P9, thereby suggesting that the pup microbiome actively contributes to immune education rather than merely reflecting immaturity. This observation aligns with prior reports that Ligilactobacillus salivarius can inhibit pathogens, modulate immune responses, and enhance intestinal barrier function [30], as evidenced by its demonstrated efficacy in alleviating Salmonella-induced mucosal damage [31]. Given the characteristically higher colonization levels of this genus in early life, we propose that its enrichment during this critical period may help shape both local and systemic immune status by modulating inflammatory signaling pathways and fine-tuning immune regulation.

Notably, Akkermansia muciniphila colonizes the intestinal mucus layer and colon, where it reinforces the mucus barrier by modulating goblet cell mucin secretion while potentially delivering anti-aging benefits through nicotinamide production [32]. Its dysregulation is involved in the pathological processes of various diseases, including diabetes, obesity, cardiovascular diseases, immune disorders, cancer, and neurological diseases [33]. In this study, the enrichment of Akkermansia during adulthood aligns with a phase of heightened metabolic demand and intestinal maturation. Given its established roles in metabolic regulation and anti-inflammation, we hypothesize that its mid-life abundance may underpin long-term health. Conversely, the decline observed in old mice likely reflects dysbiosis associated with conventional aging. Intriguingly, the sustained presence of Akkermansia in long-lived populations suggests its potential as a biomarker of healthy aging and longevity potential [34], highlighting the value of exploring dietary or probiotic strategies to maintain its abundance in later life.

The dynamic change in SCFAs, key microbial metabolites with established roles in enhancing barrier function, modulating immunity, and maintaining microbial balance [35,36,37], aligns with intestinal development across the lifespan. Consistent with previous reports, a distinct SCFA profile was observed in pups-P9, characterized by low total SCFA levels, a marked deficiency in acetate, and a high relative proportion of butyrate. This pattern likely reflects a milk-based, fiber-deficient diet, suggesting that butyrate may serve as a crucial compensatory developmental signal under these conditions, while acetate deficiency could impair intestinal barrier function and increase susceptibility to diseases such as necrotizing enterocolitis in early life [38]. With increasing age, intestinal barrier function gradually matures, and the total SCFAs reach the peak during the adult-to-middle-age stage of the life cycle. Muribaculaceae might be the major contributor in adults since it can metabolize both endogenous (mucin glycans) and exogenous (dietary fiber) polysaccharides to produce SCFAs [24], among which, Dubosiella has been reported to play an important immunomodulatory role in maintaining intestinal homeostasis and alleviating colitis [39]. In this study, we observed an enrichment of the genus Desulfovibrio in the intestines of adult mice, whose abundance was positively correlated with isobutyric acid level, traditionally perceived as a harmful one. However, Hong et al. [40] reported a positive correlation between Desulfovibrio abundance and acetate levels, linking it to a healthier metabolic state, suggesting that the functional role of this genus may be condition-dependent and potentially beneficial in specific conditions. Numerous studies have reported a decline in butyrate levels and microbial dysbiosis associated with aging [41]. Notably, our study did not observe a significant decrease in butyrate in old mice. This discrepancy may be attributed to the controlled dietary conditions, highlighting the critical role of diet in shaping gut microbiota function and metabolite output.

A key finding of this study is that aged mice display increased basal expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and TLR4 in the colon yet show markedly diminished acute inflammatory responses to IL-1β stimulation in ex vivo tissues. This coexistence of elevated inflammatory mediators with attenuated responsiveness suggests complex regulatory dynamics that could reflect immune desensitization, receptor-level regulation, or altered cellular composition [42]. During aging, the intestine is chronically exposed to low-grade inflammatory stimuli resulting from dysbiosis and impaired barrier function [43]. This chronic stimulation induces a dual adaptive reorganization of the immune system. On one hand, sustained activation of negative feedback pathways (such as chronic nuclear factor kappa-B (NF-κB) signaling upregulating inhibitors like IκBα, and prolonged mitogen-activated protein kinase (MAPK) activation inducing phosphatases) establishes an “endotoxin tolerance-like” state that prevents excessive inflammatory damage [44,45]. On the other hand, profound shifts occur in intestinal immune cell populations with a reduction in naive responder cells and an increase in the proportion of senescent and memory cells [46,47]. Collectively, this adaptive reorganization represents a “defensive compromise” by the immune system under chronic stress: persistent low-level release of inflammatory mediators under basal conditions, but a diminished capacity to mount rapid, robust responses to acute stimuli.

The PI3K/Akt pathway displayed a biphasic activation pattern, with significantly elevated activity in both pup-P9 and old groups compared to adults. In pups-P9, despite overall low total SCFA level, butyrate constitutes a relatively higher proportion. Recent studies indicate that butyrate can promote intestinal development by activating the PI3K/Akt signaling pathway, including epithelial proliferation, differentiation, and villus-crypt maturation [48,49]. Correspondingly, we observed elevated PI3K/Akt activity in pups-P9, which likely represents a compensatory response [50,51]. However, this may be insufficient to fully establish robust barrier function, resulting in heightened disease susceptibility [52]. The PI3K/Akt pathway exhibits well-established functional heterogeneity across intestinal cell types, regulating proliferation and barrier function in epithelial cells [53,54,55] while modulating activation and effector functions in immune cells [44,56,57,58]. This duality may explain the biphasic activation pattern observed in this study (elevated in both early life and old age) which likely reflects the predominant contributions of distinct cell populations at different stages. Specifically, elevated activity in early life may stem from rapidly dividing epithelial cells during intestinal maturation [44,53], whereas in old age, it may be driven by chronically stimulated immune cells and the senescence-associated secretory phenotype (SASP) [57,59]. During adulthood, sufficient SCFAs provide the gut with abundant energy substrates and signaling molecules, allowing the PI3K/Akt signaling pathway to operate at a physiologically homeostatic level of activation. This balance ensures normal renewal and metabolism of the intestinal epithelium while preventing excessive activation, thereby maintaining optimal intestinal barrier function [60]. Despite the maintenance of SCFA levels, the old group demonstrated hyperactivation of the PI3K/Akt pathway. We speculate that this reflects a pathological shift driven by the systemic, low-grade inflammatory microenvironment characteristic of aging. Such inflammation-coupled activation likely promotes cellular stress, compromises barrier function, and contributes to metabolic disorders, thereby forming a vicious cycle [61]. The preservation of SCFA levels in old age suggests that age-related functional decline may arise not from an absolute deficit in production, but rather from altered host responsiveness of the immune system.

As a key molecule connecting innate immunity and inflammatory responses, TLR4 plays a critical role in intestinal homeostasis. Appropriate TLR4 signaling supports epithelial repair and immune tolerance [14], whereas excessive activation promotes NF-κB and MAPK pathways, leading to elevated pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) that compromise barrier integrity [13]. We observed that TLR4 and these pro-inflammatory cytokines increase with age, yet the response to stimuli such as IL-1β is attenuated. This indicates a state of TLR4 dysregulation, a hallmark of immunosenescence, which directly fuels “inflammaging” [14]. A potential positive correlation was observed between pro-inflammatory genera, such as Turicibacter, and intestinal inflammation, coinciding with the age-related increase in IL-1β, IL-6, and TNF-α in old groups. This trend suggests that microbial shifts may contribute to the pro-inflammatory milieu of the aging gut, although this interpretation remains preliminary and hypothesis-generating due to the limited sample size and correlational nature of the data. This is consistent with the study of Fan et al. [62], which linked such dysbiosis to barrier disruption, systemic LPS elevation, and TLR4/NF-κB pathway activation. Interestingly, a similar positive correlation was observed with Limosilactobacillus, a genus typically considered beneficial. Although a previous report shows that Limosilactobacillus fermentum ME-3 can suppress paracellular permeability and pro-inflammatory cytokine release in LPS-induced cell models [63], its enrichment in middle-aged and old groups here suggests that the aged host environment may alter host–microbe communication, potentially converting commensal or probiotic signals into pro-inflammatory triggers [64,65]. We speculate that age-related decline in intestinal barrier function and altered immune surveillance drive these microbial and immune changes, which then engage in pathogenic crosstalk with intracellular metabolic pathways, notably the PI3K/Akt axis, collectively promoting a vicious cycle of barrier dysfunction and metabolic disturbance. The persistent TLR4 activation likely contributes to the pathological sustained activation of PI3K/Akt in aging. In turn, this hyperactive PI3K/Akt pathway may amplify inflammatory signals and inhibit repair processes, creating a vicious feedback loop that synergistically exacerbates barrier decline, metabolic disturbance, and immune dysfunction [14]. The biphasic activation of PI3K/Akt (elevated in both pups-P9 and old mice) parallels the distinct microbial configurations observed at these stages, underscoring the dynamic interplay between host signaling and the gut ecosystem. In pups-P9, this activation likely supports compensatory adaptation to low SCFA availability and immature barrier function. In old mice, sustained PI3K/Akt activation is consistent with the enrichment of pro-inflammatory taxa such as Turicibacter and the dysregulated TLR4 signaling observed in this group, suggesting that the microbiota–PI3K/Akt axis contributes to both developmental maturation and age-related inflammatory susceptibility [53]. Thus, the dysregulated TLR4-PI3K/Akt interface emerges as a critical mechanistic nexus linking microbial ecology, chronic inflammation, and the parallel phenomena of immunosenescence and inflammaging in the aging gut. It is important to note that the current study design, while revealing temporal associations, does not establish direct causality between age-related microbiota alterations and the activation of TLR4–PI3K/Akt signaling. Beyond TLR4 signaling, other innate immune pathways, including the NOD-like receptor family, pyrin domain-containing protein 3 inflammasome, cyclic GMP-AMP synthase—stimulator of interferon genes (cGAS-STING), and interferon-gamma/extracellular signal-regulated kinase/mitogen-activated protein kinase (IFN-γ/ERK/MAPK) cascades, have been implicated in intestinal aging, contributing collectively to barrier dysfunction, cellular senescence, and impaired stem cell maintenance [10,66,67]. These pathways likely do not operate in isolation; rather, future studies should investigate their potential crosstalk with the TLR4-PI3K/Akt axis. Elucidating this broader immunometabolic network will be essential for advancing a system-level understanding of age-related immune dysregulation in the gut.

5. Conclusions

This study identifies a lifespan-dependent reconfiguration of the gut ecosystem, characterized by coordinated shifts in microbial composition, SCFA profiles, TLR4 signaling, and PI3K/Akt pathway activity. Age-associated intestinal decline arises not merely from microbial compositional changes but from a fundamental, stage-specific remodeling of host–microbe interactions, with immune–metabolic crosstalk as a central feature. These findings provide a microecology-centered framework for understanding the parallel processes of immunosenescence and inflammaging. Although these findings offer a mechanistic framework relevant to the murine model, their direct applicability to humans is limited by interspecies physiological variation. Additionally, the small sample size and the lack of sex-specific analysis limit statistical power and generalizability, precluding robust assessment of sex-related variability. Given the correlational nature of the microbiome data, these findings should be considered hypothesis-generating. Larger and well-powered cohort studies are needed to further validate these observations, enable sex-specific analyses, and establish causal relationships. In parallel, future research should focus on the lifespan-wide microbiota–host signaling axis, particularly the TLR4-PI3K/Akt signaling pathway, using mechanistic interventions such as cell-specific deletions or microbiota transplantation. A deeper understanding of these dynamics may ultimately inform strategies, including life stage-tailored prebiotics or targeted modulation of host signaling pathways, to support gut health and promote healthier aging.